Factors affecting energy transfer from phycobilisomes to thylakoids in Anacystis nidulans

Short illumination with white light of dark-maintained Anacystis nidulans prior to immersion in liquid nitrogen resulted in a marked change of fluorescence emission characteristics at 77 K. The fluorescence of Photosystem II-associated membrane bound pigments increases, while the emission due to phycobilins decreases. This effect seems to be due to a light-dependent alteration in the extent of contact between phycobilisomes and thylakoids, since the effect is reversible in the dark and is abolished by short glutaraldehyde fixation. The preillumination effect is not inhibited by DCMU. Emission spectra obtained with actively growing and CO₂-starved cells indicate that the light-dependent increase in energy transfer from phycobilins to chlorophyll depends upon the physiological state of the cells.     

Energy transfer from phycobiliproteins to photosystem I in vegetative cells and heterocysts of Anabaena variabilis

The presence of phycobilins in heterocysts of Anabaena variabilis is established on the basis of absorption and fluorescence spectroscopy. At 77 K heterocysts exhibit fluorescence emission bands at 645 and 661 nm indicative of phycocyanin and allophycocyanin, respectively. Both allophycocyanin levels and fluorescence emission at 695 nm were low in heterocysts relative to whole filaments. In situ fluorescence microscopy confirmed the presence of phycobilins in individual heterocysts, but the pigment levels varied considerably among cells. Heterocysts exhibited Photosystem I activity, as evidenced by photooxidation of P-700, but no Photosystem II activity. The quantum efficiency of phycobilins in sensitizing P-700 photooxidation was 50-70% that of chlorophyll a. Phycoibins were also effective in promoting light-dependent reduction of acetylene to ethylene. The results are discussed in terms of the role of the heterocyst in nitrogen fixation and of the significance of energy transfer from phycobilins to Photosystem I in heterocysts.     

Photoprotection of reaction centers: thermal dissipation of absorbed light energy vs charge separation in lichens

During desiccation, fluorescence emission and stable light-dependent charge separation in the reaction centers (RCs) of photosystem II (PSII) declined strongly in three different lichens: in Parmelia sulcata with an alga as the photobiont, in Peltigera neckeri with a cyanobacterium and in the tripartite lichen Lobaria pulmonaria. Most of the decline of fluorescence was caused by a decrease in the quantum efficiency of fluorescence emission. It indicated the activation of photoprotective thermal energy dissipation. Photochemical activity of the RCs was retained even after complete desiccation. It led to light-dependent absorption changes and found expression in reversible increases in fluorescence or in fluorescence quenching. Lowering the temperature changed the direction of fluorescence responses in P. sulcata. The observations are interpreted to show that reversible light-induced increases in fluorescence emission in desiccated lichens indicate the functionality of the RCs of PSII. Photoprotection is achieved by the drainage of light energy to dissipating centers outside the RCs before stable charge separation can take place. Reversible quenching of fluorescence by strong illumination is suggested to indicate the conversion of the RCs from energy conserving to energy dissipating units. This permits them to avoid photoinactivation. On hydration, re-conversion occurs to energy-conserving RCs.     

Excitation-energy distribution in green algae. The existence of two independent light-driven control mechanisms

Three distinct states can be identified for cells of the green alga Chlorella vulgaris; State 1 and State 2 obtained by preillumination in far-red and red light, respectively, and the dark state obtained by dark-adaptation. Addition of the inhibitor DCMU to algal cells leads to an initial rapid increase in chlorophyll-a fluorescence reflecting the closure of Photosystem II traps. This, in the case of dark and state-2-adapted algae is followed by a slow light-dependent increase to a fluorescence yield typical of State-1-adapted cells. Measurements of low temperature (77 K) emission spectra indicate that the low fluorescence yields of dark and State-2-adapted algae reflect similar balances in excitation-energy distribution between the two photosystems. In both cases, the balance favours PS I and the slow fluorescence increase seen in the poisoned algae reflects a redressing of this balance in favour of PS II. The low fluorescence yield of State-2-adapted algae is thought to be associated with the phosphorylation of chlorophyll a/b light-harvesting protein (Biochim. Biophys. Acta (1983) 724, 94–103). Measurements of the uncoupler and ATPase sensitivity of the light-dependent increases seen in DCMU-poisoned cells indicate that the low fluorescence yield of dark-adapted algae is of different origin. Evidence is presented showing that the light-driven changes in excitation-energy distribution seen in green algae involve two distinct processes; a low-intensity, wavelenght-independent change reflecting simple light/dark changes and a higher intensity, wavelength-dependent change reflecting State 1/State 2 adaptation. The former changes appear to be associated with changes in the local ionic environment within the algal chloroplast, whilst the latter appear to reflect changes in the phosphorylation state of chlorophyll a/b light-harvesting protein.     

Membrane Development in the Cyanobacterium, Anacystis nidulans, during Recovery from Iron Starvation

Deprivation of iron from the growth medium results in physiological as well as structural changes in the unicellular cyanobacterium Anacystis nidulans R2. Important among these changes are alterations in the composition and function of the photosynthetic membranes. Room-temperature absorption spectra of iron-starved cyanobacterial cells show a chlorophyll absorption peak at 672 nanometers, 7 nanometers blue-shifted from its normal position at 679 nanometers. Iron-starved cells have decreased amounts of chlorophyll and phycobilins. Their fluorescence spectra (77K) have one prominent chlorophyll emission peak at 684 nanometers as compared to three peaks at 687, 696, and 717 nanometers from normal cells. Chlorophyll-protein analysis of iron-deprived cells indicated the absence of high molecular weight bands. Addition of iron to iron-starved cells induced a restoration process in which new components were initially synthesized and integrated into preexisting membranes; at later times, new membranes were assembled and cell division commenced. Synthesis of chlorophyll and phycocyanins started almost immediately after the addition of iron. The absorption peak slowly returned to its normal wavelength within 24 to 28 hours. The fluorescence emission spectrum at 77K changed over a period of 14 to 24 hours during which the 696- and 717-nanometer peaks grew to their normal levels, and the 684 nanometer peak moved to 687 nanometers and its relative intensity decreased to its normal level. Analysis of chlorophyll-protein complexes on polyacrylamide gels showed that high molecular weight chlorophyll-protein bands were formed during this time, and that low molecular weight bands (related to photosystem II) disappeared. The origin of the fluorescence emission at 687 and 696 nanometers is discussed in relation to the specific chlorophyll-protein complexes formed during iron reconstitution.     

Biosynthesis of phycobilins. Ferredoxin-supported nadph-independent heme oxygenase and phycobilin-forming activities from Cyanidium caldarium.

The unicellular red alga, Cyanidium caldarium, synthesizes phycocyanobilin from protoheme via biliverdin IX alpha. In vitro transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins were previously shown to require NADPH, ferredoxin, and ferredoxin-NADP+ reductase, as well as specific heme oxygenase and phycobilin formation enzymes. The role of NADPH in these reactions was investigated in this study. The C. caldarium enzymatic activities that catalyze biliverdin IX alpha formation from protoheme, and phycobilin formation from biliverdin IX alpha, were partially purified by differential (NH4)2SO4 precipitation. The enzyme fractions, when supplemented with a light-driven ferredoxin-reducing photosystem I fraction derived from spinach leaves, catalyzed light-dependent transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins, with or without the addition of NADPH and ferredoxin-NADP+ reductase. In the dark, neither reaction occurred unless NADPH and ferredoxin-NADP+ reductase were supplied. These results indicate that the only role of NADPH in both reactions of phycobilin biosynthesis, in vitro, is to reduce ferredoxin via ferredoxin-NADP+ reductase and that reduced ferredoxin can directly supply the electrons needed to drive both steps in the transformation of protoheme to phycocyanobilin.     

THE PHYCOBILIN SIGNATURES OF CHLOROPLASTS FROM THREE DINOFLAGELLATE SPECIES: A MICROANALYTICAL STUDY OF DINOPHYSIS CAUDATA, D. FORTII, AND D. ACUMINATA (DINOPHYSIALES, DINOPHYCEAE)

The absorbance and fluorescence emission spectra for three species of Dinophysis, D. caudata Saville-Kent, D. fortii Pavillard, and D. acuminata Claparède et Lachmann, were obtained through an in vivo microanalytical technique using a new type of transparent filter. The pigment signatures of these Dinophysis species were compared to those of Synechococcus Nägeli, a cryptophyte, and two wild rhodophytes, as well as those of another dinoflagellate, a diatom, and a chlorophyte. Phycobilins are not considered a native protein group for dinoflagellates, yet the absorption and fluorescence properties of the three Dinophysis species were demonstrated to closely resemble phycobilins and chlorophylls of Rhodomonas Karsten (Cryptophyceae). Analyses of Dinophysis species using epifluorescence microscopy found no additional nucleus or nuclear remnant as would be contributed by an endosymbiont.     

Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua: Analysis of Fluorescence Kinetics as a Function of Excitation Intensity

The chlorophyll fluorescence induction curves from mesophyll and guard cell chloroplasts of Saxifraga cernua, including both the fast (O to P, the transients involved in the rise in variable fluorescence) and slow (P to steady state fluorescence due to quenching) components, were characterized over a range of excitation intensities using microspectrophotometry (with epi-lumination) equipped with apertures designed to eliminate cross contamination of the fluorescence signal between the two chloroplast types. At low excitation intensities, the fast fluorescence kinetics from guard cell plastids showed an extended I to D phase and a more rapid appearance of P while minimal quenching from P to steady state fluorescence was observed compared to the transients from mesophyll chloroplasts suggesting a lower activity of photochemical (electron movement via carriers between donor and acceptor sites) and nonphotochemical (such as membrane conformational changes) events which regulate the fluorescence induction curve kinetics. As the excitation intensity was increased, the quenching rates of guard cells were faster at initiating conditions for photophosphorylation and the fast and slow fluorescence kinetics from guard cells resembled those of the mesophyll cells.

Guard cell chloroplasts of S. cernua from intact epidermal peels showed a low temperature (77 K) fluorescence emission spectrum having three major peaks (at 685, 695, and 730 nanometers when excited at 440 nanometers) which were qualitatively similar to those in the spectrum obtained from mesophyll tissue.

These data suggest that S. cernua guard cell chloroplast photosystems I and II contribute to light-dependent stomatal activity only at high light intensities.

     

Photoprotection of green plants: a mechanism of ultra-fast thermal energy dissipation in desiccated lichens

In order to survive sunlight in the absence of water, desiccation-tolerant green plants need to be protected against photooxidation. During drying of the chlorolichen Cladonia rangiformis and the cyanolichen Peltigera neckeri, chlorophyll fluorescence decreased and stable light-dependent charge separation in reaction centers of the photosynthetic apparatus was lost. The presence of light during desiccation increased loss of fluorescence in the chlorolichen more than that in the cyanolichen. Heating of desiccated Cladonia thalli, but not of Peltigera thalli, increased fluorescence emission more after the lichen had been dried in the light than after drying in darkness. Activation of zeaxanthin-dependent energy dissipation by protonation of the PsbS protein of thylakoid membranes was not responsible for the increased loss of chlorophyll fluorescence by the chlorolichen during drying in the light. Glutaraldehyde inhibited loss of chlorophyll fluorescence during drying. Desiccation-induced loss of chlorophyll fluorescence and of light-dependent charge separation are interpreted to indicate activation of a highly effective mechanism of photoprotection in the lichens. Activation is based on desiccation-induced conformational changes of a pigment-protein complex. Absorbed light energy is converted into heat within a picosecond or femtosecond time domain. When present during desiccation, light interacts with the structural changes of the protein providing increased photoprotection. Energy dissipation is inactivated and structural changes are reversed when water becomes available again. Reversibility of ultra-fast thermal dissipation of light energy avoids photo-damage in the absence of water and facilitates the use of light for photosynthesis almost as soon as water becomes available.     

Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the powerful genetically encoded tag presently available in a variety of mutants featuring blue to yellow emission, has found a red-emitting counterpart. The recently cloned red fluorescent protein DsRed, isolated from Discosoma corals (), with its emission maximum at 583 nm, appears to be the long awaited tool for multi-color applications in fluorescence-based biological research. Studying the emission dynamics of DsRed by fluorescence correlation spectroscopy (FCS), it can be verified that this protein exhibits strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities and excitation wavelengths suggest that DsRed appears under equilibrated conditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induces transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensity. With increasing intensity, the emission maximum of the static fluorescence continuously shifts to the red, implying that at least one state emitting at longer wavelength is preferably populated at higher light levels. In close resemblance to GFP, this light-induced dynamic behavior implies that the chromophore is subject to conformational rearrangements upon population of the excited state.     

Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.     

Nitrogen fixation associated with a hotspring green alga

A unicellular alga which can grow in the light without a combined nitrogen source was isolated from a hot spring. The cells were almost spherical, usually 5–10 m in diameter. Absorption spectra of the watersoluble pigments and of the acetone-extracted ones revealed the existence of chlorophyll a and b and the absence of phycobilins. Thin sections examined by electron microscopy revealed an eukaryotic organization with features typical of the coccoid green algae (the Chlorococcales). Cells divided by internal cytokinesis and subsequent liberation of daughter cells from the parental wall, in a way similar to Chlorella. The alga reduced acetylene to ethylene and incorporated ¹⁵N₂ into cell protoplasm when incubated in a low oxygen atmosphere. Nitrogenase activity was light-dependent, microaerophilic and thermophilic. Although the association of symbiotic nitrogen fixing prokaryotes with the cells may still be possible, any such organisms have not so far been detected.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Chl chlorophyll - MBM modified Bristol medium - TLC thin layer chromatography     

Light-dependent proton transport into mesophyll vacuoles of leaves of C3 plants as revealed by pH-indicating fluorescent dyes: a reappraisal

Esculin, a pH-sensitive fluorescent dye, was used to indicate light-dependent pH changes in leaves of Spinacia oleracea L. and Pelargonium zonale L. Shortly after its introduction into the leaves via the transpiration stream, esculin was localized mainly in the symplasm. An increase in its blue fluorescence on illumination with red actinic light indicated that the cytosolic pH had increased. A similar light-dependent alkalinization was seen when the green fluorescence of pyranine was used to monitor changes in the cytosolic pH. After esculin had been transferred into the vacuoles, a light-dependent vacuolar acidification was indicated by a decrease in its blue fluorescence. Since the pK of esculin is close to neutrality, it is suitable as an indicator of proton transport into vacuoles provided the vacuolar sap is only moderately acidic. In leaf cells with very acidic vacuoles, esculin therefore responds only to cytosolic pH changes as long as it remains in the cytosol. The observations made with esculin after it had entered the vacuoles confirmed earlier conclusions on light-dependent proton transport into the vacuoles of mesophyll cells. Previous measurements had been made with 5-carboxy-2,7-dichlorofluoresceine (CDCF), which has a pK of 4.8. In contrast to esculin, CDCF can, in principle, record pH changes in very acidic vacuoles. However, earlier conclusions made on the basis of observed CDCF fluorescence are now recognized to have no unambiguous basis because new measurements, reported here, show that CDCF fluorescence is influenced not only by pH changes but also by changes in light scattering. The latter are, like pH changes, light-dependent and originate from the thylakoid system of chloroplasts. They indicate both the formation of a large transthylakoid proton gradient and the dissipation of excess light energy as heat. Decreased green fluorescence of leaves which had been fed CDCF may therefore, depending on conditions, indicate vacuolar acidification or the dissipation of excess light energy absorbed by the pigment system of chloroplasts, or both. Pyranine fluorescence was found to be much less influenced by light scattering than CDCF fluorescence.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluoresceine - P₇₀₀ primary donor of PS I - PFD photon flux density - Q_A primary quinone acceptor of PS II - Q_P, Q_N photochemical, non-photochemical quenching of chlorophyll fluorescence, respectively This work was supported by the Deutsche Forschungsgemeinschaft within the framework of the research of the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful to Drs. U. Schreiber and K.-J. Dietz and to Mrs. B. Hollenbach (all from our Institute) for discussions.     

Cyanobacterial thylakoid membrane proteins are reversibly phosphorylated under plastoquinone-reducing conditions in vitro

Reversible, light-dependent protein phosphorylation was observed in isolated thylakoid membranes of the cyanobacterium Synechococcus 6301. A polypeptide of 15 kDa in particular was phosphorylated under plastoquinone-reducing conditions and was not phosphorylated under plastoquinone-oxidising conditions. Phosphorylation and dephosphorylation reactions involving this and several other membrane polypeptides showed sensitivity to inhibitors of protein kinases and phosphatases. Changes in phosphorylation state correlated with changes in low temperature fluorescence emission characteristic of changes in excitation energy distribution between the photosystems. The 15 kDa phosphopolypeptide is likely to be involved directly in light state adaptations in cyanobacteria.     

The photoreduction of H(2)O(2) by Synechococcus sp. PCC 7942 and UTEX 625

It has been claimed that the sole H(2)O(2)-scavenging system in the cyanobacterium Synechococcus sp. PCC 7942 is a cytosolic catalase-peroxidase. We have measured in vivo activity of a light-dependent peroxidase in Synechococcus sp. PCC 7942 and UTEX 625. The addition of small amounts of H(2)O(2) (2.5 microM) to illuminated cells caused photochemical quenching (qP) of chlorophyll fluorescence that was relieved as the H(2)O(2) was consumed. The qP was maximal at about 50 microM H(2)O(2) with a Michaelis constant of about 7 microM. The H(2)O(2)-dependent qP strongly indicates that photoreduction can be involved in H(2)O(2) decomposition. Catalase-peroxidase activity was found to be almost completely inhibited by 10 microM NH(2)OH with no inhibition of the H(2)O(2)-dependent qP, which actually increased, presumably due to the light-dependent reaction now being the only route for H(2)O(2)-decomposition. When (18)O-labeled H(2)O(2) was presented to cells in the light there was an evolution of (16)O(2), indicative of H(2)(16)O oxidation by PS 2 and formation of photoreductant. In the dark (18)O(2) was evolved from added H(2)(18)O(2) as expected for decomposition by the catalase-peroxidase. This evolution was completely blocked by NH(2)OH, whereas the light-dependent evolution of (16)O(2) during H(2)(18)O(2) decomposition was unaffected.     

Effects of solar and artificial radiation on motility and pigmentation in Cyanophora paradoxa

The effects of solar radiation and artificial UV irradiation on motility and pigmentation were studied in the flagellate system Cyanophora paradoxa. Both percentage of motile cells and average velocity decreased drastically after a solar exposure of a few hours. This effect was not due to an overheating since the cells were exposed under temperaturecontrolled conditions. Partial reduction of the UV-B radiation by cut-off filters or by insertion of an artificial ozone layer increased the tolerated exposure times. Artificial UV radiation also induced the same effects. Under both solar and artificial UV irradiation the photosynthetic pigments within the cyanelles were bleached also within short exposure times. Kinetics of pigment destruction showed that the accessory phycobilins are lost with a half life of 1.3 h while the chlorophylls had a half life of 33 h and a carotenoid with an absorption maximum at 480 nm of 17.3 h.     

STRESS‐INDUCED FILAMENT FRAGMENTATION OF CALOTHRIX ELENKINII (CYANOBACTERIA) IS FACILITATED BY DEATH OF HIGH‐FLUORESCENCE CELLS1

Irradiance power and spectral composition as well as nutrient availability strongly influence differentiation of filamentous cyanobacteria. When monitoring the life cycle of Calothrix elenkinii Kossinsk., we found that low nitrogen concentration and growth under green light led to a transient appearance of high‐fluorescence cells that rapidly bleach and disintegrate, thus breaking the parental filament into shorter parts. The dynamics of the process were monitored in a microscope growth chamber by measuring transmission and chl fluorescence of individual cells by a high‐sensitivity camera. Typically, the high‐fluorescence cells appeared near the center of the parental trichome signaled by a rapid 2‐ to 3‐fold rise in their fluorescence emission. By measuring the fluorescence excitation spectra with resolution of individual cells, we showed that the elevated fluorescence emission was largely due to a high absorption by phycoerythrin and energy transfer to chl. Typically, after no more than 20 min, the fluorescence abruptly disappeared with transmission images, indicating loss of pigmentation. The bleaching was a natural process that was not caused by the measuring light. Depending on the mechanical strain, the cell bleaching was followed by breaking of the parental filament. We propose that the high‐fluorescence cells appear as a phase of programmed cell death, allowing the fragmented filaments to escape from unfavorable environmental conditions.     

Fluorescence emission spectra of cells and subcellular preparations of a green photosynthetic bacterium. Effects of dithionite on the intensity of the emission bands

Fluorescence emission spectra were measured of intact cells and subcellular preparations of the green photosynthetic bacterium Prosthecochloris aestuarii in the presence and in the absence of dithionite. A 3–5-fold increase in bacteriochlorophyll a fluorescence at 816 nm occurred upon addition of dithionite in a membrane vesicle preparation (Complex I), in a photochemically active pigment-protein complex and in a bacteriochlorophyll a protein complex free from reaction centers. The pigment-protein complex showed a relatively strong long-wave emission band (835 nm) of bacteriochlorophyll a, which was preferentially excited by light absorbed at 670 nm and was not stimulated by dithionite. With Complex I, which contains some bacteriochlorophyll c in addition to bacteriochlorophyll a, a 3–4-fold stimulation of bacteriochlorophyll c emission was also observed. Emission bands at shorter wavelengths, probably due to artefacts, were quenched by dithionite. With intact cells, the effect of dithionite was smaller, and consisted mainly of an increase of bacteriochlorophyll a emission.

The results indicate that the strong increase in the yield of bacteriochlorophyll emission that occurred upon generating reducing conditions is, at least mainly, due to a direct effect on the light-harvesting systems, and does not involve the reaction center as had been earlier postulated.     

Flow-cytometric analysis of chicken red blood cells.

Flow-cytometric analysis of acriflavin-Feulgen stained chicken erythrocytes shows a complex distribution of amounts of deoxyribonucleic acid fluorescence, the profile consisting of a main peak and a right hand shoulder. This bimodal distribution, an artifact characteristically seen on analysis of flattened cells using orthogonal flow systems, results from fluorescence emission in preferred directions stemming from the combined effects of refractility and orientation of the cells. The shoulder disappears on analysis of lysed erythrocyte ghosts, also on analysis of cells in a medium whose refractive index approximates that the cells. An orientation effect for matrue erythrocytes was indicated by reanalysis of fractions after sorting on the basis of high and low fluorescence or scatter signals. Both fractions gave the original range of values on reanalysis, although some changes in shape of the profile and in the peak positions for the sorted cells were seen. Sodium dodecyl sulfate treatment of stained cells "loosened" the cells' structure, yielding lowered scatter values, and fluorescence values approaching those of the shoulder. The average fluorescence emission of the erythrocytes was lower than that of reticulocytes and lymphocytes. The values of the latter correspond closely, although coincidently, to that the erythrocyte shoulder values. Dual parameter analysis of forward light scatter, and fluorescence, which was detected at 90 degrees to the laser beam, showed the low fluorescence to be accompanied by low scatter signal, and the high fluorescence among the cells with the high scatter signal. The lowered forward scatter signal is due to a wider scattering of light from cells oriented edge-on to the detector, and loss of signal beyond the acceptance angle of the detector. These results suggest that the preferred directions for fluorescence are in the plane of the cells, and the values are dependent on the cells' orientation in the stream. These interpretations were supported by the results of analysis of partially oriented cells. The approaches used and conclusions arrived at are similar to those of Gledhill et al (16), Van Dilla et al (37), in their analysis of fluorescence of flat sperm cells although the affects in the case of the erythrocytes are less extreme.     

Drying pretreatment enhances the photosynthetic stability of alginate immobilisedPhormidium laminosum

Summary Immobilisation of the thermophilic cyanobacteriumP. laminosum in alginate beads with a drying pretreatment led to stabilization of the Photosystem II water splitting activity for at least 3 months. This was accompanied by an increase of energy transfer from phycobilins, a maintenance of the variable fluorescence and a red shift of 77k emissions of allophycocyanin and PSI complexes. These changes indicate improvements in the photosynthetic efficiencies of immobilised cells.Abbreviations PSI, II Photosystem I, II: Chl a: chlorophyll a - LHC light harvesting complex - DCP IP 2–6 dichlorophenol indophenol - DCMU 3(3,4-dichlorophenyl)-1,1 dimethylurea - IMPD 2, 3, 5, 6 tetramethyl phenylenediamine