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Temperature dependence of chlorophyll a fluorescence in relation to the physical phase of membrane lipids algae and higher plants.

The temperature dependence of the yield of chlorophyll a fluorescence was measured at room temperatures in living algal cells and higher plant chloroplasts. 3-(3',4'-Dichlorophenyl)-1, 1-dimethylurea was added to the samples during the measurements in order to eliminate the influence of photosynthetic photochemical reactions on the fluorescence yield.In Anacystis nidulans the maximum in the curve for the fluorescence yield versus temperature occurred near the temperatures at which the transition of physical phase of the membrane lipids changes from the liquid crystalline to the mixed solid-liquid crystalline states. These findings suggest that the occurrence of a fluorescence maximum in the temperature-dependence curve is an indication of the thermal transition of physical phase of membrane lipids. Similar but less distinct maxima were found in Cyanidium caldarium and Euglena gracilis. No maxima were seen in the curves for chlorophyll a fluorescence versus temperature in Chlorella pyrenoidosa, Plectonema boryanum, Stichococcus bacillaris Phaeodactylum tricornutum, or in chloroplasts of the higher plants, spinach, lettuce, tomato, and Tidestromia oblongifolia.The fluorescence yield of allophycocyanin in Anacystis did not show such a maximum, but did show a steep increase with decreasing temperature below 5 C. the fluorescence yields of chlorophyll a and phycobilins extracted from Anacystis increased monotonously with decreasing temperature.

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Polypeptide composition of chlorophyll-protein complexes from romaine lettuce.

The protein moiety of the two major chlorophyll-protein complexes associated with chloroplast membranes of outer, dark green leaves of a romaine lettuce shoot (Lactuca sativa L. var. Romana) has been analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complex II, also termed light-harvesting chlorophyll-protein complex, is shown to consist of a major polypeptide of 25 kilodaltons (kD) and two minor ones of 27.5 and 23 kD. The 25 kD subunit is the single largest polypeptide component of the chloroplast membranes, accounting for about 25% of their total protein. Complex I contains only high molecular weight subunits, the major one being at 67 kD, these subunits representing only a small percentage of the chloroplast membrane total protein.These data, suggesting an oligomeric nature for the apoprotein of these two chlorophyll-protein complexes, are difficult to reconcile with the estimated molecular weights of the native complexes and raise some intriguing questions as to the types of interactions among the components of these major lipoproteins of the photosynthetic membranes.

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Comparison of Light-dependent Oxygen Uptake, Protochlorophyll(ide)-650 Photoconversion, and Chlorophyll Disappearance in Wheat Etioplasts.

Red light exposures given to dark-grown wheat seedlings (Triticum aestivum L.) prior to etioplast isolation reduced the ability of these organelles to consume O(2). The same preharvest red light exposures also decreased protochlorophyll(ide) content of etioplasts. In addition, regeneration of both O(2) uptake rates as well as protochlorophyll(ide) levels followed a parallel time course. These similarities suggested that photoconversion of protochlorophyll(ide)-650 to chlorophyll(ide) may mediate some process with O(2) as the electron acceptor. This process appears to involve photooxidation of nonphotoconvertible protochlorophyll(ide) as well as of newly formed chlorophyll(ide). This hypothesis is further supported by the observations that: (a) the in vitro light induced O(2) uptake phenomenon was observed in solubilized protochlorophyll(ide) holochrome preparations; and (b) photoinduced O(2) uptake was reduced to zero rate by light exposure time equivalent to that required for chlorophyll(ide) and nonphotoconvertible protochlorophyll(ide) destruction.

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Azolla-Anabaena Relationships: VII. Distribution of Ammonia-assimilating Enzymes, Protein, and Chlorophyll between Host and Symbiont.

The N(2)-fixing Azolla-Anabaena symbiotic association is characterized in regard to individual host and symbiont contributions to its total chlorophyll, protein, and levels of ammonia-assimilating enzymes. The phycocyanin content of the association and the isolated blue-green algal symbiont was used as a standard for this characterization. Phycocyanin was measured by absorption and fluorescence emission spectroscopy. The phycocyanin content and total phycobilin complement of the symbiotic algae were distinct from those of Anabaena cylindrica and a free-living isolate of the Azolla endophyte. The algal symbiont accounted for less than 20% of the association's chlorophyll and protein. Acetylene reduction rates in the association (based solely on the amount of algal chlorophyll) were 30 to 50% higher than those attained when the symbiont was isolated directly from the fern. More than 75% of the association's glutamate dehydrogenase and glutamine synthetase activities are contributed by the host plant. The specific activity of glutamate dehydrogenase is greater than that of glutamine synthetase in the association and individual partners. Both the host and symbiont have glutamate synthase activity. The net distribution of these enzymes is discussed in regard to the probable roles of the host and symbiont in the assimilation of ammonia resulting from N(2) fixation by the symbiont.

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Analysis of the Light-harvesting Pigment-Protein Complex of Wild Type and a Chlorophyll-b-less Mutant of Barley.

we have compared chloroplast lamellae isolated from a chlorophyll-b-less mutant and wild type barley (Hordeum vulgare). The results demonstrate that: (a) one of the two major polypeptides comprising the lightharvesting complex (LHC) is present in the chlorophyll-b-less mutant; (b) higher cation concentrations are required to maintain grana stacks in the mutant; and (c) cation effects on excitation energy distribution are present in the chlorophyll-b-less mutant but are reduced in amount and are dependent on higher concentrations of cations.We interpret these data to support the concept that the LHC mediates cation-induced grana stacking and cation regulation of excitation energy distribution between photosystems I and Ii in chloroplast lamellae. A partial LHC complement in the mutant alters the quantitative cation requirement for both phenomena, but not the over-all qualitative response.

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Photoactivation of Chlorophyll Synthesis and Cytochrome Oxidase Activity in Anaerobically Germinated Seedlings of Echinochloa crusgalli var. Oryzicola.

When seeds of Echinochloa crusgalli var. oryzicola are germinated in dark anaerobic conditions (99.995% N(2)), the seedlings do not have detectable protochlorophyll(ide). Two hours after exposure to light aerobic conditions, they begin to synthesize chlorophyll. The lag in greening is shorter in seedlings exposed to light for 24 hours before exposure to air. Seedlings maintained in light anaerobic conditions exhibit no lag in greening upon transfer to an aerobic environment. Preillumination of anaerobically grown seedlings does not result in any chlorophyll accumulation. Phytochrome is probably the receptor for photoactivation of chlorophyll synthesis, since activation is achieved by red light alone, but not by far red light or red plus far red light. The cytochrome oxidase activity in anaerobically germinated seedlings is 30% of the normal level found in aerobically grown seedlings. Preillumination was also found to activate the ability of anaerobically germinated seedlings to increase their cytochrome oxidase activity upon exposure to air.

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Influence of Cell Age on Chlorophyll Formation in Light-grown and Etiolated Wheat Seedlings.

A method is described for relating the age of a cereal leaf cell to its distance from the leaf base. The rates of chlorophyll synthesis per plastid in the first leaf of light-grown and of greening etiolated seedlings of wheat (Triticum aestivum, var. Maris Dove) increase with cell age. Normally developing plastids of light-grown wheat take over 24 hours to reach the chlorophyll a/b ratio characteristic of mature wheat chloroplasts (4.5), but mature etioplasts need only 8 hours light to achieve this a/b ratio. Plastid greening potential depends only on cell age, whereas the chlorophyll a/b ratio is influenced both by cell age and by light.

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Functional and Structural Organization of Chlorophyll in the Developing Photosynthetic Membranes of Euglena gracilis Z: II. CHARACTERISTICS OF THE LATE FORMATION OF ACTIVE PHOTOSYSTEM II REACTION CENTERS DURING FIRST STAGES OF GREENING.

During light-induced greening of dark-grown, nondividing Euglena gracilis Z, there is a delay of about 10 hours in the formation of active photosystem II (PSII) reaction centers compared to chlorophyll synthesis. Experiments with greening under different light intensities rule out the possibility that this delay results from a late induction of active PSII reaction center formation when a definite amount of chlorophyll is attained in the early greened cells. Experiments on greening after preillumination show that this delay does not originate in a long, light-induced formation of specific synthesizing machinery for reaction center components. Experiments with greening in the presence of streptomycin show that, when this inhibitor of protein synthesis by chloroplastic ribosomes is added to dark-grown, preilluminated cells or to cells already greened for 24 hours, the formation of active PSII reaction centers is inhibited after a time which depends on the light intensity used for greening. Under very low light intensity (150 lux), the addition of streptomycin to 24-hour greened cells does not prevent further development of functional chloroplasts. These observations lead to the conclusion that streptomycin-insensitive chloro-plast development occurs due to syntheses of cytoplasmic origin and of light-induced pools of components synthesized early by chloroplastic ribo-somes. Conformational changes requiring time may allow the insertion of components necessary for the reorganization of PSII reaction centers in the developing thylakoid after synthesis. This hypothesis accounts for the observed delay in PSII reaction center formation compared to chlorophyll synthesis.

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Coupling of chlorophyll metabolism with submembrane chloroplast particles, isolated with digitonin and gel electrophoresis.

An unusual set of submembrane particles is obtained from digitonintreated barley chloroplasts as five gel-electrophoretic zones. Four of them are photochemically active, whereas the most mobile fifth zone has essential traits of the light-harvesting complexes. All of the particles contain the well-known chlorophyll-protein complexes and represent an intermediate level of membrane organization. When isolated from plants fed delta-aminolevulinate in the dark, the fifth zone is characterized by a high level of protochlorophyllide, which is also present to a lesser extent in all the other zones. When [(14)C]aminolevulinate was fed in the dark, followed by exposing the plants to light, the same pattern of the distribution was observed for [(14)C]chlorophyll a. Thus, particles of all the types are involved in chlorophyll formation and the fifth zone is the most distinct in this respect. Its material seems to originate from the most intensely developing areas of the metabolically heterogeneous chloroplast membrane system.

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Evidence of singlet oxygen participation in the chlorophyll-sensitized photooxidation of indoleacetic Acid.

Chlorophyll-sensitized photooxidation of indoleacetic acid (IAA)-with chlorophyll extracted from Pisum sativum L. cv. Alaska W.R.-was determined in the presence of deuterium oxide and known quenchers of singlet oxygen ((1)O(2)) to explore the involvement of (1)O(2) in the reaction. O(2) uptake was measured in light in a buffered aqueous micellar system containing Triton X-100, KCl, chlorophyll, and IAA. The rate of O(2) uptake was zero in darkness. The reaction was stimulated by deuterium oxide and inhibited by sodium azide indicating that (1)O(2) participated in IAA photooxidation. Both mannitol and superoxide dismutase failed to inhibit O(2) uptake suggesting that neither the hydroxyl radical nor the superoxide anion played a significant role in the reaction.

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Effects of low concentrations of o(3) on net photosynthesis, dark respiration, and chlorophyll contents in aging hybrid poplar leaves.

Chronic exposure of hybrid poplar (Populus deltoides x trichocarpa) plants to low concentrations of ozone had negative impact upon net photosynthetic capacity, dark respiration, and leaf chlorophyll contents. Exposure to as much as 0.20 microliters per liter O(3) had no immediate effect on net photosynthesis (P(n)), but chronic exposure to 0.125 or 0.085 microliters per liter had a number of gradual effects on CO(2) exchange. These included increased dark respiration and consequently increased light compensation points in very young leaves (4-6 days old); and decreased P(n), leaf chlorophyll a and b contents, light saturation points, and apparent quantum yields in leaves 10 to 70 days old. Decreased P(n) was partially due to accelerated aging in leaves exposed to O(3), and lightsaturated P(n) was linearly related to chlorophyll a + b contents. Differences in light-saturated P(n) between control and O(3)-treated leaves of the same age were mostly due to photosaturation in O(3)-treated leaves and to a much lesser extent to lowered apparent quantum yields. Also, since P(n) and dark respiration were most affected by O(3) at different leaf ages, distinct modes of action are suggested. The effects of leaf aging on CO(2) exchange were considerable, but typical of other species. However, careful monitoring of the interacting effects of leaf age and pollutant exposure was needed in order to characterize the impact of chronic O(3) exposure upon CO(2) exchange.

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Induction of the De Novo Formation of the Photosystem I-Related Light-Harvesting Chlorophyll Protein Complex LHCPa by Photoheterotrophic Nutrition.

The green alga Chlamydobotrys stellata contains in addition to the normal light-harvesting chlorophyll protein complex LHCPb a special LHCPa which is free of chlorophyll b and connected only to photosystem I (Brandt, Zufall, Wiessner 1983 Plant Physiol 71: 128-131). The kinetics of these two LHCP forms were analyzed during the transition in nutrition of the alga from autotrophy to photoheterotrophy, e.g. the replacement of CO(2) by acetate as carbon source. As shown by incorporation experiments with [(14)C]acetate, this change in nutrition leads to an increased synthesis of LHCPa, whereas the synthesis of the photosystem II-related LHCPb decreases. The increase of the LHCPa synthesis starts immediately after the onset of photoheterotrophic nutrition together with the synthesis of the chlorophyll protein complex CPI. There is no interchange of complex components between LHCPa and LHCPb during the depletion of the latter. The formation of LHCPa is discussed with respect to the regulation of gene expression.

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Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, and Cytochrome b(6)/f Complex.

Changes in the amount of P700-chlorophyll a protein complex, plastocyanin, and cytochrome b(6)/f complex during greening of pea (Pisum sativum L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) leaves were analyzed by an immunochemical quantification method. Neither subunit I nor II of P700-chlorophyll a protein complex could be detected in the etiolated seedlings of all three plants and the accumulation of these subunits was shown to be light dependent. On the other hand, a small amount of plastocyanin was present in the etiolated seedlings of all three plants and its level increased about 30-fold during the subsequent 72-hour greening period. Furthermore, cytochrome f, cytochrome b(6), and Rieske Fe-S center protein in cytochrome b(6)/f complex were also present in the etiolated seedings of all three plants. The level of each subunit component increased differently during greening and their induction pattern differed from species to species. The accumulation of cytochrome b(6)/f complex was most profoundly affected by light in pea leaves, and the levels of cytochrome f, cytochrome b(6), and Rieske Fe-S center protein increased during greening about 10-, 20-, and more than 30-fold, respectively. In comparison to the case of pea seedlings, in wheat and barley leaves the level of each subunit component increased much less markedly. The results suggest that light regulates the accumulation of not only the chlorophyll protein complex but also the components of the electron transport systems.

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Functional Organization of Chlorophyll a and Carotenoids in the Alga, Nannochloropsis salina.

Chlorophyll-protein complexes were isolated from a yellow-green alga, Nannochloropsis salina after mild detergent treatment and gel electrophoresis. Three different complexes were obtained which correspond to the three major kinds of chlorophyll-proteins isolated from spinach chloroplasts by the same procedure and previously identified as reaction center complexes for photosystems I and II and a light-harvesting complex. The analogy between the algal complexes and those from spinach was drawn from their absorption and fluorescence spectra and relative pigment content. The identities and amounts of the major carotenoids associated with each isolated complex were determined by HPLC. Although the reaction center complexes accounted for only 14% of the total chlorophyll, they were highly enriched in beta-carotene, whereas the light-harvesting complex contained a high proportion of xanthophylls (mainly violaxanthin and vaucheriaxanthin-ester). Fluorescence excitation spectra of the algal membranes showed that one or both of the major xanthophylls may act as antenna pigment for photosynthesis.

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Gabaculine Inhibition of Chlorophyll Biosynthesis and Nodulation in Phaseolus lunatus L.

Gabaculine (3-amino-2,3-dihydrobenzoic acid) was an inhibitor of in vivo chlorophyll biosynthesis in lima bean (Phaseolus lunatus L. cv Henderson). When applied to roots of 9-day-old plants, 10 micromolar gabaculine was sufficient to terminate biosynthesis of new chlorophyll. The trifoliolate leaves which emerged after gabaculine treatment were yellow. Gabaculine-treated plants had slightly lower dry weights; yet, overall plant size showed very little change. Chlorophyll fluorescence induction kinetics and CO(2) exchange measurements were used to monitor both immediate and long-term effects of gabaculine on photosynthesis. A lowered rate of the decline from the maximum level of fluorescence was observed after 10 hours for nitrate-supplemented plants, and all treated plants showed a slightly increased level of original fluorescence after 6 days. No change was observed in the rate of photosynthesis by unifoliolate leaves. The trifoliolate leaves, though not able to photosynthesize, were able to continue respiration. This suggested that heme biosynthesis for mitochondrial cytochromes was not abolished. In untreated lima bean, root nodules were induced by Rhizobium sp. 127E15. Following gabaculine treatment, root nodules formed, but were largely ineffective in nitrogen fixation. Nodule dry weight, nitrogen fixation activity, and leghemoglobin content were decreased by gabaculine.

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Phytochrome regulation of greening in barley-effects on chlorophyll accumulation.

Red light treatment of dark-grown 6-day-old barley seedlings (Hordeum vulgare L.) strongly reduces the lag in chlorophyll accumulation in subsequent white light over that found in dark control seedlings placed under white light. Fluence-response studies show that the effect has both very low fluence and low fluence components. Kinetic studies indicate that the reduction in lag begins immediately following either a low fluence or a very low fluence red irradiation, with the initial rate of change significantly lower after the very low fluence treatment and showing sharp far red-absorbing form of phytochrome dependence. In both cases, the effect is maximal after roughly 4 hours, either remaining fairly constant (very low fluence) or declining somewhat (low fluence) thereafter. Saturating far red light alone yields a response equivalent to very low fluence red, and will reverse only the low fluence component of the red response. Escape from far red reversibility occurs gradually over about a 3 hour period. Since the kinetics described here differ from those in the literature related to phytochrome effects on transcription of the mRNA for the light-harvesting chlorophyll a/b-binding protein, we conclude that the phytochrome-regulated component of chlorophyll accumulation is not limited by transcription of the mRNA for its major apoprotein. Leaf segments vacuum-infiltrated with water retain the capacity to green in white light. If they are infiltrated with mannitol solutions of various concentrations, their capacity to green declines sharply at concentrations above 0.2 molar. These results bear on interpretation of run-on transcription experiments with isolated nuclei: preparation of the nuclei involves enzymic digestion of the tissue in the presence of 0.7 molar mannitol for 2.5 hours, to obtain protoplasts prior to breaking the cells. The results here make it unlikely that normal transcriptional regulation is occurring during this procedure.

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Chlorophyll a Fluorescence Yield as a Monitor of Both Active CO(2) and HCO(3) Transport by the Cyanobacterium Synechococcus UTEX 625.

Simultaneous measurements have been made of inorganic carbon accumulation (by mass spectrometry) and chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625. The accumulation of inorganic carbon by the cells was accompanied by a substantial quenching of chlorophyll a fluorescence. The quenching occurred even when CO(2) fixation was inhibited by iodoacetamide and whether the accumulation of inorganic carbon resulted from either active CO(2) or HCO(3) (-) transport. Measurement of chlorophyll a fluorescence yield of cyanobacteria may prove to be a rapid and convenient means of screening for mutants of inorganic carbon accumulation.

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Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates.

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.

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