Intact Algae Research Articles

Nutritional Analysis of Puget Sound Bull Kelp (Nereocystis luetkeana)

ABSTRACTSamples of Nereocystis luetkeana, an edible brown algae, were collected from Puget Sound, Wash. The freeze‐dried samples were analyzed for proximate and elemental composition and evaluated for protein quality by amino acid analysis and using Tetrahymena pyriformis W. The freeze‐dried frond, stipe, and bulb of the algae showed similar composition; however, the frond had a protein content twice that found in the bulb and stipe and five times less crude fiber than the stipe. The proximate analysis of the frond was 15.3% protein, 1.3% crude fiber, 42.7% ash, 1.9% crude fat, and 38.8% carbohydrate (by difference). Based on neutron activation analysis, Nereocysti: contained appreciable quantities of sodium, potassium, calcium, and iron. The algae protein appeared to be of high quality based on growth of Tetrahymena pyriformis W. on a pepsin digest of the algae and on the amino acid profile and in vitro digestibility of the intact algae.

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Measurements of ribulose‐1,5‐bisphosphate carboxylase (RUBPCase) and phosphoenolpyruvate carboxylase (PEPCase) in several phytoplankton cultures and natural phytoplankton populations showed a positive correlation of photosynthesis with RUBPCase activity but not with PEPCase activity. Observed ratios of RUBPCase:PEPCase were high in log phase cultures when photosynthesis was maximal and low in stationary phase. There was no significant correlation between photosynthesis and the ratio of RUBPCase:PEPCase activity in natural populations. The rate of carbon assimilation by the intact algae was significantly greater than that by the isolated carboxylases. In both natural phytoplankton populations and laboratory cultures, the ratio of photosynthesis:carboxylase activity depended on the physiological state of the cells, being greatest when the photosynthetic rate was highest.

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part I. In the intact alga

The wavelength-resolved fluorescence emission kinetics of the accessory pigments and chlorophyll a in Porphyridium cruentum have been studied by picosecond laser spectroscopy. Direct excitation of the pigment B-phycoerythrin with a 530 nm, 6 ps pulse produced fluorescence emission from all of the pigments as a result of energy transfer between the pigments to the reaction centre of Photosystem II. The emission from B-phycoerythrin at 576 nm follows a nonexponential decay law with a mean fluorescence lifetime of 70 ps, whereas the fluorescence from R-phycocyanin (640 nm), allophycocyanin (660 nm) and chlorophyll a (685 nm) all appeared to follow an exponential decay law with lifetimes of 90 ps, 118 ps and 175 ps respectively. Upon closure of the Photosystem II reaction centres with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination the chlorophyll a decay became non-exponential, having a long component with an apparent lifetime of 840 ps. The fluorescence from the latter three pigments all showed finite risetimes to the maximum emission intensity of 12 ps for R-phycocyanin, 24 ps for allophycocyanin and 50 ps for chlorophyll a. A kinetic analysis of these results indicates that energy transfer between the pigments is at least 99% efficient and is governed by an exp −At 1 2 transfer function. The apparent exponential behaviour of the fluorescence decay functions of the latter three pigments is shown to be a direct result of the energy transfer kinetics, as are the observed risetimes in the fluorescence emissions.

Functional characteristics of intact chloroplasts isolated from mesophyll protoplasts and bundle sheath cells of maize

A procedure was developed to isolate mesophyll and bundle sheath chloroplasts of a high degree of intactness and low cross-contamination. Light-induced (14)CO2 fixation of isolated chloroplasts was similar to that of protoplasts and cells in that it was low and was stimulated by the addition of exogenous substrates. O2 evolution was absent in both bundle sheath chloroplasts and cells. The flash-induced 515 nm absorbance change of intact mesophyll chloroplasts showed a biphasic rise, previously known to be a characteristic only of intact algae. With bundle sheath chloroplasts or cells, no 515 nm signal could be detected. In the presence of 10 μmol l(-1) phenazine methosulphate, bundle sheath chloroplasts exhibited a flash-induced 515 nm signal with a monophasic rise and amplitude comparable to that of the mesophyll chloroplasts. A similar signal was obtained with bundle sheath chloroplasts suspended in an extract prepared from the mesophyll tissue. Both the substrate stimulation of the CO2 fixation and the reconstitution of the 515 nm signal in bundle sheath chloroplasts by the mesophyll extract indicate the requirement of cooperation between the mesophyll and bundle sheath cells of maize leaves.

Photosynthetic carboxylating enzymes in Phaeodactylum tricornutum: Assay methods and properties

Rapid freezing (in liquid nitrogen) of the marine diatom Phaeodactylum tricornutum Bohlin followed by thawing permits a convenient and sensitive measurement of the activities of carboxylating enzymes without the need to prepare a cell-free extract. Using this method, the properties of RuDP and PEP carboxylases have been compared with those assayed in cell-free extracts. The most significant difference was in the Michaelis' constants (K m 's); the values being lower in the freeze/thaw assay. The absolute rate of carbon-dioxide fixation by the enzymes was less than the rate of photosynthesis by the intact alga. Significantly, the activity of PEP carboxylase was comparable (in some experiments, greater) to that of RuDP carboxylase. The significance of this and the possibility of an enzymatic approach to measurements of marine primary productivity are discussed.

Antagonistic relationships between electron transport and P700 in chloroplasts and intact algae.

The effects of divalent salts and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) upon nicotinamide adenine dinucleotide phosphate (NADP) reduction and P700 in isolated chloroplasts are described and compared with the influence of DCMU on oxygen evolution and P700 in intact cells. Most experiments were carried out with a steady-state relaxation spectrometer. A kinetic mechanism for the estimation of P700 fluxes is proposed and experimentally tested. Good agreement between theory and experiment was found. Concurrent measurements of P700 and NADP reduction revealed two antagonisms: addition of divalent cations caused an increase in the yield of NADP reduction and a decrease in the yield of P700. Conversely, in the presence of Mg2+ low concentrations of DCMU decreased the yield of NADP reduction and increased the yield of P700. Aging of the chloroplasts at 30 degrees C exerted a similar effect. With far-red actinic light, Mg2+ stimulated the yield of NADP reduction without affecting the flux. Also, in the absence of Mg2+, DCMU inhibited both reactions although P700 required a higher herbicide concentration for fractional inhibition than NADP reduction. In the presence of Mg2+, chloroplasts resembled intact algae in which a high rate of oxygen evolution was accompanied by little P700 turn-over. Titration with DCMU decreased the rate of photosynthesis and increased P700 flux. On the whole, the data suggest that P700 relaxing in 20 msec is not directly involved in linear electron transport.

Chlorophyllase and Lamellar Structure in Phaeodactylum tricornutum: I. Chlorophyll → Chlorophyllide Conversion Within the Lamellae

1. Chlorophyllase in the marine diatom Phaeodactylum tricornutum is inactive, or nearly so, in vivo, but becomes active upon desintegration of the cell structure by osmotic shock or French Press treatment. Cell desintegration is apparent from disappearance of long-wave chlorophyll forms (685 and 710 nm), present in the intact algae (Goedheer, 1973). 2. Chlorophyllase activity can be measured down to -10°C. 3. No correlation was found between the relative amount of the combined long-wave chlorophyll forms in the absorption spectrum of the intact cells and chlorophyllase activity measured in cells which had received an osmotic shock. 4. In the presence of Mg2+ (0.01 M) osmotic shock of the cells results in a lower chlorophyllase activity than that which is found upon the same treatment without Mg2+. The absorption difference spectrum of cells, treated in the presence and absence of Mg2+, shows a maximum at about 685 nm. 5. Treatment of the osmotically shocked cells with oleic acid (ratio oleic acid/ chlorophyll 8 : 1) increases chlorophyllase activity. Absorption at 685 nm has decreased as compared with that of cells not treated with oleic acid. 6. It is concluded that chlorophyll in the lamellae is in vivo protected from chlorophyllase, but becomes exposed to the enzyme upon cell desintegration. The disappearance of the 685 nm chlorophyll form is suggested to be quantitatively related to this effect.

Chloroplast Functions are Influenced by Morphactins

Summary Labelled morphactins become associated with the chloroplasts of Acetabularia. The binding of labelled indol acetic acid is hindered by a pretreatment of the algae with morphactins (20 μg/ml). The photosyntheic rate is decreased and the circadian pattern of oxygen evolution is altered by morphactins (20 μg/ml). Morphactins (2 and 20 μg/ml) bring about an increase in the chloroplast polysaccharide content of the intact, but not of the anurleate Acetabularia. Morphactins (2 and 20 μg/ml) induce an intrease in uridine incorporation into the acid-insoluble fraction of Acetabularia in the light in intact algae and in algae anucleated since one week. Morphactins thus act on chloroplast activities in several ways. The results are discussed and the effects of morphactins and those of anucleation are compared.

Light-regulation of enzyme activity in anacystis nidulans (Richt.)

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

EPR signals of oxidized plastocyanin in intact algae

1. 1. An electron paramagnetic resonance (EPR) signal was observed at g = 2.05 in the low temperature spectra of intact cells of green, red and blue-green algae and of spinach chloroplasts. The g-value and the shape of the signal were similar to that of purified, soluble plastocyanin. 2. 2. The amount of the copper protein, determined from the EPR signal height, was estimated to be nearly the same in all the studied organisms on the basis of the concentration of chlorophyll. Furthermore, it was found that the amount of the copper protein, determined from the EPR signal height in spinach chloroplasts corresponds with that of plastocyanin as determined chemically by Katoh, S., Suga, I., Shiratori, I. and Takamiya, A. (1961) Arch. Biochem. Biophys. 94, 136–141. 3. 3. Experiments with far-red and red illumination show that the site of the copper protein in vivo is in the electron transport pathway between Photosystems 1 and 2. Plastocyanin is not oxidized by illumination at 77 °K, indicating that no electron transfer occurs between the primary electron donor of Photosystem 1, P700, and plastocyanin at that temperature. Furthermore, the experiments suggest that in the intact cells of the studied algae, plastocyanin is not only reduced by Photosystem 2 but also by cyclic electron transport around Photosystem 1.

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

The luciferin-luciferase method was used to determine ATP extracted from darkmaintained and light-exposed samples of the green alga Chlorella pyrenoidosa and of the blue-green alga Anacystis nidulans. A few measurements on Synechococcus lividus (a bluegreen thermophile, clone 65°C) are also reported. The light-minus-dark ATP levels (δATP) from aerobic cells of Chlorella and Anacystis were negative; however, δATP from Synechococcus was positive. Large positive δATP was obtained in regularly grown (RG: moderate light) Chlorella treated with oligomycin; dark levels were reduced, light levels remained essentially unaffected. In high-light exposed (HLE) Chlorella, oligomycin reduced both light and dark ATP levels, but positive δ ATP was still obtained. However, in Anacystis, which has a different organization of thylakoid membrane, oligomycin severely reduced both the light and the dark ATP levels and the δATP remained negative. The oligomycin (12 μM) treated Chlorella and the untreated Anacystis and Synechococcus show the presence of cyclic photophosphorylation under conditions in which the non-cyclic electron flow from photosystem 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. Cyclic photophosphorylation ranged from 10–30% of the maximum δATP in RG, to 40–50% in HLE Chlorella. In RG Chlorella, cyclic and non-cyclic (in the absence of DCMU) photophosphorylation (δATP) saturate at about 103 ergs cm−2 sec−1 and 104 ergs cm−2 sec−1 and 104 ergs cm−2 sec−1 red (>640 nm) light, respectively; a lag was observed in the light curve. In Chlorella, the addition of the photosystem I electron acceptor methyl viologen (MV; 1 mM) increased δATP by twofold. Further addition of DCMU (25 μm) reduced this to the level observed with DCMU alone. If 1 mM reduced dichlorophenol indophenol or phenazine methosulphate (DCPIPH2 or PMSH2, respectively) was added along with DCMU, the δATP level was 30–40% of the control. Further addition of MV increased the JATP to be 70–80% of that of the control. These and other results confirm the presence of both non-cyclic and cyclic photophosphorylation in vivo, the former predominating in Chlorella, and the latter in Anacystis and Synechococcus.

Symbiosis in sacoglossan opisthobranchs: photosynthetic products of animal-chloroplast associations

Alcohol-soluble photosynthetic products of chloroplasts symbiotic in three species of sacoglossan slugs (Elysia hedgpethi, Placida dendritica Placobranchus ianthobapsus) were analyzed and compared to products of chloroplasts in an intact alga. Animal-cell chloroplasts appear to lack the ability to synthesize either lipids or sucrose. They do, however, produce greater percentages of TCA-cycle intermediates than do chloroplasts from the intact alga investigated.

Photooxidation of 3,3′-diaminobenzidine by blue-green algae and Chlamydomonas reinhardii

1. 1. The photooxidation of 3,3′-diaminobenzidine was investigated in whole cells of the wild-type and two mutant strains of Chlamydomonas reinhardii and in four species of blue-green algae. 2. 2. In the wild-type strain, this reaction is insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and is dependent on the presence of methyl viologen. The photooxidation can be demonstrated in a mutant strain lacking Photosystem II activity. However, it is absent from a mutant strain deficient in Photosystem I activity. 3. 3. Experiments performed with chloroplast fragments isolated from the wildtype and mutant strains yielded results similar to those obtained with whole cells. These results established that the photooxidation of 3,3′-diaminobenzidine is mediated by Photosystem I only. 4. 4. In contrast to Chlamydomonas reinhardii, blue-green algae are able to photooxidize 3-3′-diaminobenzidine at high rates in the absence of methyl viologen. This property is attributed to the ability of the primary electron acceptor of Photosystem I to undergo autooxidation in vivo. 5. 5. The possible applications of this reaction in the studies of photosynthetic electron transport reactions in intact algae and in the investigations of chloroplast substructures are discussed.

Reaction kinetics of intermediates of the photosynthetic chain between the two photosystems

1. 1. Measurements are reported of light-induced changes in absorbance and in the yield of chlorophyll a fluorescence of intact algae. Most experiments were done with the red alga Porphyridium aerugineum. In alternating non-saturating illumination with light mainly absorbed by Photosystem 1 and 2, respectively, a periodical oxidation and reduction of the components of the photosynthetic chain, P700, cytochrome f, plastoquinone and Q, the primary electron acceptor of Photosystem 2, was observed. 2. 2. Lags observed in the oxidation and reduction kinetics agreed with the concept that the midpoint potentials of these compounds decrease in the order given above. The largest difference was observed between plastoquinone and cytochrome f; the kinetics of Q and plastoquinone were similar and suggested only a small potential difference between these compounds. 3. 3. Relative pool sizes of 15:1.8:1 electron equivalent were observed for plastoquinone, cytochrome f and P700. Quantum efficiencies suggested the same high rate of electron flow through plastoquinone and cytochrome f. The kinetics of these components indicated that the only large pool in the chain between the two light reactions is that of plastoquinone. 4. 4. Difference spectra obtained under conditions where all the above-mentioned components were alternately reduced and oxidized gave no evidence for cytochrome b participation in the chain between the two light reactions.

The Influence of Two Naturally Occurring Factors, from the Leaves of Ricinus Communis, on the Fluorescence Induction of Higher Plant Chloroplasts and Intact Algae

Changes in fluorescence induction, brought about by incubation of chloroplasts (Zea mays) in an aqueous extract of Ricinus leaf, have been divided, on the basis of speed of manifestation, into two categories: "fast" changes and "slow" changes (i.e. those observed after 5 min and 1(1/2) hr of incubation, respectively). The former, which include a large increase in the magnitude of the fast component of variable fluorescence and a retardation of decay from maximum to minimum levels of fluorescence, have been ascribed to inhibition of electron transport at a site beyond that of 3-(p-chlorophenyl)-1,1-dimethylurea (CMU)-i.e., towards system I; these changes result from the action of a fraction of the extract consisting of molecules of small size. The latter changes, which include a marked attenuation of the variable part of fluorescence induction, have been associated with system II and may arise from inhibition of electron flow between water and Q or from decrease in number of functional reaction centers; these changes result from the activity of a proteinaceous fraction of the extract, that simultaneously converts the low temperature steady-state emission spectrum of the chloroplasts into a one-banded one, with maximum at 698 nm.

GNOTOBIOTIC CULTURE AND PHYSIOLOGICAL ECOLOGY OF THE MARINE NEMATODE RHABDITIS MARINA BASTIAN1

The marine nematode Rhabditis marina Bastian has been isolated in gnotobiotic culture from collections of Zostera marina made in North Sea Harbor, Southampton, New York. A strain of R. marina has been maintained with three species of bacteria (Pseudomonas sp., Flavobacterium marinum, and Micrococcus sp.) for over 120 generations. A monoxenic strain with Pseudomonas sp. has been maintained for over 25 generations. The life cycle is completed in 5 days under “normal” laboratory growth conditions. Reproduction occurs over a wide (0–80‰) salinity range (the highest salinity tested) and a wide (10–38C) temperature range; optimum growth occurs at 45–55‰ and 25–30C. Normal development is oviparous, but ovoviviparous development is seen in older females. Tracer feeding and synxenic culture experiments have shown that R. marina has rather specific nutritional requirements, which seem to be completely satisfied by a relatively restricted group of microorganisms. Selected species of bacteria comprise the normal diet of this organism. Microscopic observations of intact algae in the digestive tract of the worms and failure of 32P to transfer to the worms from the ingested algae indicate that the worms are not able to digest all ingested potential food organisms.The geographical distribution of the species in relation to its physiological ecology is discussed.

Cation effects on the fluorescence of isolated chloroplasts.

At room temperature, the fluorescence of chloroplasts in vivo and in vitro originates nearly exclusively from the chlorophyll of the oxygen evolving system II of the photosynthetic apparatus. The yield and the kinetics of this light emission are closely coupled to the primary photochemical events of photosystem II. Fluorescence studies, therefore, have contributed much to our knowledge about the water oxidizing photoact. According to Duysens and Sweers i(3) the fluorescence yield is determined by the oxidation state of the primary electron acceptor Q: when Q is oxidized, the fluorescence is quenched and, consequently, a low yield is observed; since reduced Q1(Q-) does not quench, a maximal fluorescence yield indicates a complete reduction of the electron acceptor pools containing Q. Several anomalies in the fluorescence kinetics of intact algae have led to the assumption of an non-homogeneous pool Q containing, even after the primary activation step (8), active and inactive oxidants Qa and Qi' (1,2, 4,12). Both forms of Q are thought to quench the fluorescence in their oxidized states, albeit with slightly different effectiveness. A photoreduction of the total pool Q requires an activation of any Qi' to Qa, which according to some authors (1,4,12) is sensitized by photosystem I. In this communication, we wish to report on an overriding influence of the ionic environment on the fluorescence yield of isolated chloroplasts. It will be shown that the fluorescence yield can be changed at will simply by varying the composition of the suspension medium. Accordingly, the fluorescence yield may possibly be governed not only by the oxidation state olf the Q-pool, but also by ion induced structural changes.

Feeding Behaviour of Cod (Gadus morhua)

Cod 5–90 cm long in captivity used sight to detect food in midwater. Movement of food stimulated feeding but was not essential when feeding on familiar foods. Adult cod could detect and capture falling food down to 2 mm in diameter. Large pieces of food on the bottom were found by sight but smaller pieces were taken after being detected by taste buds on the trailing barbel and pelvic fin rays. Cod were able to detect odours from many live, intact algae, invertebrates, and fish. Food below gravel or stones was located by smell and uncovered by carrying away gravel in the mouth or by rolling stones aside with the head. Food covered by sand was not found. Some live food was captured in the dark. Cod groups fed more effectively than single cod as food deeply buried was uncovered by several fish digging in the same place; food too large for one fish was pulled apart between fish and feeding behaviour by one fish attracted others to the vicinity of food. Thus shoaling of cod may be advantageous to the species by enabling more food to be obtained from a given area.Larval cod reared from eggs first fed 4 days after hatching. Moving particles were followed by eye movements and captured by swimming forward and snapping if the particles moved in front of the head. Eight days after hatching the cod larvae began to feed on fine stationary particles by removing them from the respiratory water flow.

Quenching of chlorophyll fluorescence by quinones in algae and chloroplasts

1. A number of quinones and substituted quinones quenched strongly chlorophyll fluorescence in Swiss chard chloroplasts and in the intact algae, Ulva lobata and Porphyra perforata. Quenching occurred in the absence as well as in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea. 2. Among the quinones found to have a high quenching activity were 2,3,5,6-tetramethylbenzoquinone, 2-methyl-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, phenanthrenequinone, and 1,2-dihydroxyanthraquinone. Reduced quinones had no or little quenching activity. 3. The quinones tested quenched less strongly the initial fluorescence, observed immediately upon illumination, than the subsequent rise of fluorescence yield during illumination. For the most active compounds, the concentrations needed for 50% quenching of the initial and the subsequent increase of fluorescence were about 70 and 15 μM, respectively. 4. The kinetics of fluorescence quenching at different light intensities and concentrations of quencher and absence of stimulation of O 2 evolution indicate that the quenchers do not stimulate photosynthetic electron transport but interact directly with chlorophyll molecules of photosystem 2 by formation of traps for the excitation energy. 5. In agreement with the assumption that the site of action of the quinones is near system 2, a number of these compounds inhibited light-induced cytochrome reduction in Porphyra in vivo. However, for most compounds no quantitative relation was found between the extent of inhibition of cytochrome reduction or of the Hill reaction in chloroplasts and the activity in quenching chlorophyll fluorescence.

The role of the nucleus in the circadian rhythms of Acetabularia mediterranea.

The role played by the nucleus in the circadian rhythms in Acetabularia mediterranea was investigated: these rhythms were in photosynthetic capacity and in chloroplast shape. The following results were obtained. 1. 1. Anucleate Acetabularia are able to maintain their rhythm of photosynthetvc capacity and to restore this rhythm after it has been abolished by physical means. 2. 2. The same conclusion is true for the rhythm in chloroplast shape. 3. 3. Actinomycin D dramatically inhibits the rhythm in photosynthetic capacity in intact algae. The rhythm is strongly decreased within 6 days by a concentration of 2.7 μg/ml; at a concentration of 0.27 μg/ml for 12–16 days the rhythm is abolished or very strongly reduced, depending on the experiment. 4. 4. Actinomycin D also prevents the rhythmic variation in chloroplast shape with the time period of the cycle, at the same concentration and for the same length of treatment as used for the inhibition of the rhythm in photosynthetic capacity. 5. 5. Actinomycin D does not affect the rhythm in photosynthetic capacity in anucleate algae at the concentrations used in the experiments with intact Acetabularia. 6. 6. No difference could be detected in the behaviour of anucleate basal fragments and anucleate apical fragments in the presence of actinomycin D. 7. 7. Actinomycin D does not affect the rhythm of variation in shape in chloroplasts in anucleate algae. 8. 8. The two circadian rhythms, affecting photosynthetic capacity and chloroplast shape, were always correlated. 9. 9. Ribonuclease induces an increase in the rhythm of photosynthetic capacity when the nucleated algae are returned to normal sea-water; on the contrary, the rhythm is not restored with photosynthesis when the treated algae are anucleate. The results are discussed on a molecular basis and a tentative interpretation is proposed, compatible with the present experiments and with results in the literature.