Isolation of membrane bound light-harvesting-complexes from the dinoflagellates Heterocapsa pygmaea and Prorocentrum minimum.

We have isolated Chl a-Chl c-carotenoid binding proteins from the dinoflagellates Prorocentrum minimum and Heterocapsa pygmaea grown under high (500 µmol m(-2) s(-1), HL) and low (35 µmol m(-2) s(-1), LL) light conditions. We compared various isolation procedures of membrane bound light harvesting complexes (LHCs) and assayed the functionality of the solubilized proteins by determining the energy transfer efficiency from the accessory pigments to Chl a by means of fluorescence excitation spectra. The identity of the newly isolated protein-complexes were confirmed by immunological cross-reactions with antibodies raised against the previously described membrane bound Chl a-c proteins (Boczar et al. (1980) FEBS Lett 120: 243-247). Spectroscopic analysis demonstrated the relatedness of these proteins with the recently described Chl-a-c 2-peridinin (ACP) binding protein (Hiller et al. (1993) Photochem Photobiol 57: 125-131; Iglesias Prieto et al. (1993) Phil Trans R Soc London B 338: 381-392). The water-soluble peridinin-Chl a binding-protein (PCP) was not detectable in P. minimum. Two functional forms of ACP with different pigmentation were isolated. A variant of ACP which was isolated from high-light grown cells, that specifically binds increased amounts of diadinoxanthin was compared to the previously described ACPs that bind proportionately more peridinin.

High abundance of Archaea in Antarctic marine picoplankton.

Archaea (archaebacteria) constitute one of the three major evolutionary lineages of life on Earth. Previously these prokaryotes were thought to predominate in only a few unusual and disparate niches, characterized by hypersaline, extremely hot, or strictly anoxic conditions. Recently, novel (uncultivated) phylotypes of Archaea have been detected in coastal and subsurface marine waters, but their abundance, distribution, physiology and ecology remain largely undescribed. Here we report exceptionally high archaeal abundance in frigid marine surface waters of Antarctica. Pelagic Archaea constituted up to 34% of the prokaryotic biomass in coastal Antarctic surface waters, and they were also abundant in a variety of other cold, pelagic marine environments. Because they can make up a significant fraction of picoplankton biomass in the vast habitats encompassed by cold and deep marine waters, these pelagic Archaea represent an unexpectedly abundant component of the Earth's biota.

Characterization of two full-length cDNA sequences encoding for apoproteins of peridinin-chlorophyll a-protein (PCP) complexes.

Characterizations are presented for RNA, 2 cDNA libraries, and 2 full-length cDNA sequences encoding for photosynthetic light-harvesting peridinin-chlorophyll a-protein (PCP) in the dinoflagellate Heterocapsa pygmaea. Subsequent analyses of the PCP system also indicate that (1) it is represented by multiple nuclear encoded genes, (2) a subset of mRNAs encoding for PCP apoproteins are regulated by growth irradiance, (3) PCP preproteins are larger than the mature apoproteins, and (4) PCP cDNA clones sequenced thus far contain a conserved region but are not identical. Results are discussed in the context of photoadaptation in dinoflagellates.

Calibration of an integrating sphere for determining the absorption coefficient of scattering suspensions.

Measuring the absolute absorption of suspensions of absorbing particles with unknown scattering characteristics is not possible in conventional spectrophotometers or in integrating spheres that have the sample located outside the sphere. A method for the calibration and use of an integrating sphere with a centrally located sample to measure absolute absorption coefficients of scattering suspensions is presented. Under the tested conditions the integrating sphere used in this study was insensitive to changes in the scattering coefficient of the sample but had a nonlinear response to increasing absorption of the sample, which could be corrected with an empirically derived function. This response was analyzed by using a Monte Carlo simulation, and results indicated that amplification of the absorption signal was primarily due to photons reflected from the sphere surface and the baffle reentering the cuvette. The calibration procedure described here may be generally applicable to spheres of different configurati n. An example of the use of the sphere for determining the absorption and scattering coefficients of marine phytoplankton samples is presented.

Ozone depletion: ultraviolet radiation and phytoplankton biology in antarctic waters.

The springtime stratospheric ozone (O3) layer over the Antarctic is thinning by as much as 50 percent, resulting in increased midultraviolet (UVB) radiation reaching the surface of the Southern Ocean. There is concern that phytoplankton communities confined to near-surface waters of the marginal ice zone will be harmed by increased UVB irradiance penetrating the ocean surface, thereby altering the dynamics of Antarctic marine ecosystems. Results from a 6-week cruise (Icecolors) in the marginal ice zone of the Bellingshausen Sea in austral spring of 1990 indicated that as the O3 layer thinned: (i) sea surface- and depth-dependent ratios of UVB irradiance (280 to 320 nanometers) to total irradiance (280 to 700 nanometers) increased and (ii) UVB inhibition of photosynthesis increased. These and other Icecolors findings suggest that O3-dependent shifts of in-water spectral irradiances alter the balance of spectrally dependent phytoplankton processes, including photoinhibition, photoreactivation, photoprotection, and photosynthesis. A minimum 6 to 12 percent reduction in primary production associated with O3 depletion was estimated for the duration of the cruise.

Light Regulation of Peridinin-Chlorophyll a-Protein (PCP) Complexes in the Dinoflagellate, Glenodinium sp. : Use of Anti-Pcp Antibodies to Detect Pcp Gene Products in Cells Grown in Different Light Conditions.

As a step toward developing the tools needed to study the molecular bases of light regulation of gene expression in dinoflagellates, light-harvesting peridinin-chlorophyll a-protein (PCP) complexes from Glenodinium sp. were purified and used to generate anti-PCP antibodies. Affinity purified anti-PCP antibodies were isolated from the crude anti-PCP antiserum resulting in improved specificity of immune reactions. The affinity purified anti-PCP antibodies were shown to react strongly and specifically with all major isoforms of PCP complexes in Glenodinium sp. cells, and were used to assess qualitative changes in the levels of PCP gene products in cells grown under different light conditions. Western blot analysis revealed a two- to three-fold increase in detectable PCP apoprotein in low light compared to high light grown cells. In vitro translation reactions supplied with total RNA from high and low light grown Glenodinium sp. cultures also showed an approximate twofold increase in translatable PCP mRNAs in low light grown cells as determined by immunoprecipitation of the primary translation products with affinity purified anti-PCP antibodies. In addition, PCP apoproteins appear to be encoded as larger pre-proteins, since the major immunoprecipitated products from in vitro translation are 23 and 22 kilodaltons, while mature PCP apoproteins are 15.5 kilodaltons. The parallel increases in PCP apoprotein and translatable PCP mRNAs indicate that light regulation of PCP complexes occurs at the level of PCP mRNA abundance.

Chlorophyll-Protein Complexes from the Red-Tide Dinoflagellate, Gonyaulax polyedra Stein : Isolation, Characterization, and the Effect of Growth Irradiance on Chlorophyll Distribution.

A comparision of high (330 microeinsteins per meter squared per second) and low (80 microeinsteins per meter squared per second) light grown Gonyaulax polyedra indicated a change in the distribution of chlorophyll a, chlorophyll c(2), and peridinin among detergent-soluble chlorophyll-protein complexes. Thylakoid fractions were prepared by sonication and centrifugation. Chlorophyll-protein complexes were solubilized from the membranes with sodium dodecyl sulfate and resolved by Deriphat electrophoresis. Low light cells yielded five distinct chlorophyll-protein complexes (I to V), while only four (I' to IV') were evident in preparations of high light cells. Both high molecular weight complexes I and I' were dominated by chlorophyll a absorption and associated with minor amounts of chlorophyll c. Both complexes II and II' were chlorophyll a-chlorophyll c(2)-protein complexes devoid of peridinin and unique to dinoflagellates. The chlorophyll a:c(2) molar ratio of both complexes was 1:3, indicating significant chlorophyll c enrichment over thylakoid membrane chlorophyll a:c ratios of 1.8 to 2:1. Low light complex III differed from all other high or low light complexes in that it possessed peridinin and had a chlorophyll a:c(2) ratio of 1:1. Low light complexes IV and V and high light complexes III' and IV' were spectrally similar, had high chlorophyll a:c(2) ratios (4:1), and were associated with peridinin. The effects of growth irradiance on the composition of chlorophyll-protein complexes in Gonyaulax polyedra differed from those described for other chlorophyll c-containing plant species.

Changes in Photosystem II Account for the Circadian Rhythm in Photosynthesis in Gonyaulax polyedra.

Cell-free extracts that show activity in photosynthetic electron flow have been prepared from the unicellular dinoflagellate, Gonyaulax polyedra. Electron flow, as O(2) uptake, was measured through both photo-system I and II from water to methyl viologen, through photosystem I alone from reduced 2,6-dichlorophenol indophenol to methyl viologen which does not include the plastoquinone pool or from duroquinol to methyl viologen which includes the plastoquinone pool. Electron flow principally through photosystem II was measured from water to diaminodurene and ferricyanide, as O(2) evolution. Cultures of Gonyaulax were grown on a 12-hour light:12 hour dark cycle to late log phase, then transferred to constant light at the beginning of a light period. After 3 days, measurements of electron flow were made at the maximum and minimum of the photosynthetic rhythm, as determined from measurements of the rhythm of bioluminescence. Photosynthesis was also measured in whole cells, either as (14)C fixation or O(2) evolution. Electron flow through both photosystems and through photosystem II alone were clearly rhythmic, while electron flow through photosystem I, including or excluding the plastoquinone pool, was constant with time in the circadian cycle. Thus, only changes in photosystem II account for the photosynthesis rhythm in Gonyaulax.

Photosynthetic characteristics and organization of chlorophyll in marine dinoflagellates.

The photosystem I reaction center complex, the P-700-chlorophyll a-protein, has been isolated from the photosynthetic membranes of two marine dinoflagellates, Gonyaulax polyedra and Glenodinium sp., by detergent solubilization with Triton X-100. The complexes isolated from the two species were indistinguishable, exhibiting identical absorption properties (400-700 nm) at both room (300 K) and low (77 K) temperature. The room temperature, red wavelength maximum was at 675 nm. The absorption properties, kinetics of photobleaching, sodium dodecyl sulfate electrophoretic mobilities, and chlorophyll a/P-700 ratio (50 +/- 10) of the P-700-chlorophyll a-protein complexes from the two species also were essentially the same and similar to those properties characterizing P-700-chlorophyll a-protein complexes of higher plants and green algae.Photosynthetic unit sizes were determined for cells grown at 1000 muW/cm(2). Both dinoflagellates had unit sizes (total chlorophyll/P-700 ratios) of about 600, even though the distribution of chlorophyll a, chlorophyll c, and peridinin in the light-harvesting components differed in Gonyaulax and Glenodinium. The number of photosynthetic units per cell in the two species correlates directly with their photosynthetic activities. A model is presented for the distribution of chlorophyll in the photosynthetic apparatus of these dinoflagellates which accounts for the known role of the isolated pigment-protein complexes and for the known photoadaptive physiology in pigmentation and photosynthesis for these species.

Characterization of photosynthetic rhythms in marine dinoflagellates: I. Pigmentation, photosynthetic capacity and respiration.

Circadian rhythms in photosynthesis were defined for the first time in the dinoflagellates Glenodinium sp. (M. Bernard strain) and Ceratium furca Ehrenberg (B. Meeson strain) and compared with that in Gonyaulax polyedra Stein. All three phytoplankton species had photosynthetic rhythms with daily amplitudes ranging from 3 to 5 and maxima displayed about midday. The photosynthetic pigment content and absorption properties of the cells were constant over the circadian cycle. Diurnal periodicities in respiration never accounted for more than 30% of the photosynthetic rhythm and did not persist under constant conditions. There was sufficient similarity between the circadian rhythms of these three dinoflagellates to suggest the mechanism of regulation may be the same for each of them.

Characterization of Photosynthetic Rhythms in Marine Dinoflagellates: II. Photosynthesis-Irradiance Curves and in Vivo Chlorophyll a Fluorescence.

Using data from light-dark cultures of Gonyaulax polyedra entrained to a 24-hour cycle, whole cell absorption curves and photosynthesis-irradiance curves were constructed for various circadian times. While whole cell absorbance and half-saturation constants of photosynthesis showed no statistical difference that could be directly related to the photosynthetic rhythm, the initial slope of the photosynthesis-irradiance curve was a time-dependent parameter which altered in direct proportion to the change in photosynthetic capacity. The results indicated a temporal change in the relative quantum yield of photosynthesis, and the circadian rhythmicity of light-limited photosynthesis was established under constant conditions. Circadian rhythmicity was detected in room temperature chlorophyll fluorescence yield. Low temperature fluorescence kinetics also showed fluctuations. The results suggest that regulation of photosynthesis by the biological clock of Gonyaulax may be mediated through the membrane-bound light reactions and a partial explanation of the underlying mechanism is proposed.

Molecular topology of the photosynthetic light-harvesting pigment complex, peridinin-chlorophyll a-protein, from marine dinoflagellates.

The photosynthetic light-harvesting complex, peridinin-chlorophyll a-protein, was isolated from several marine dinoflagellates including Glenodinium sp. by Sephadex and ion-exchange chromatography. The carotenoid (peridinin)-chlorophyll a ratio in the complex is estimated to be 4:1. The fluorescence excitation spectrum of the complex indicates that energy absorbed by the carotenoid is transferred to the chlorophyll a molecule with 100% efficiency. Fluorescence lifetime measurements indicate that the energy transfer is much faster than fluorescence emission from chlorophyll a. The four peridinin molecules within the complex appear to form two allowed exciton bands which split the main absorption band of the carotenoid into two circular dichronic bands (with negative ellipticity band at 538 nm and positive band at 463 nm in the case of peridinin-chlorophyl a-protein complex from Glenodinium sp.). The fluorescence polarization of chlorophyll a in the complex at 200 K is about 0.1 in both circular dichroic excitation bands of the carotenoid chromophore. From these circular dichroic and fluorescence polarization data, a possible molecular arrangement of the four peridinin and chlorophyll molecules has been deduced for the complex. The structure of the complex deduced is also consistent with the magnitude of the exciton spliting (ca. greater than 3000 cm-1) at the intermolecular distance in the dimer pair of peridinin (ca. 12 A). This structural feature accounts for the efficient light-harvesting process of dinoflagellates as the exciton interaction lengthens the lifetime of peridinin (radiative) and the complex topology increases the energy transfer probability. The complex is, therefore, a useful molecular model for elucidating the mechanism and efficiency of solar energy conversion in vivo as well as in vitro.

The role of peridinin-chlorophyll a-proteins in the photosynthetic light adaption of the marine dinoflagellate, Glenodinium sp.

The marine dinoflagellate, Glenodinium sp., was cultured at a series of light levels and growth, pigmentation, and photosynthetic rates were compared. Under decreasing light conditions, growth rates decreased, cellular chlorophyll a and peridinin content per cell increased, and maximum cellular photosynthetic rates remained unchanged. Pigmentation changes were related to alterations in cellular concentrations of a peridinin-chlorophyll a-protein and an unidentified chlorophyll a component of the chloroplast membrane. Maintenance of photosynthetic rates with decreased irradiance is interpreted as an increase in the number of pigment molecules in the light-harvesting antenna associated with the reaction centers of the photosynthetic apparatus, thus increasing the potential for photon capture for photosynthesis.

Effects of growth irradiance on the photosynthetic action spectra of the marine dinoflagellate, Glenodinium sp.

Whole cell absorption curves of the marine dinoflagellate Glenodinium sp., cultured at irradiances of 250µW/cm(2) (low light) and 2500µW/cm(2) (high light), were measured and their difference spectrum determined. Absorption by low light grown cells exceeded that of high light grown cells throughout the visible spectrum by a factor which ranged from 2 to 4. The difference spectrum supported the view that increased pigmentation, resulting from low light conditions, was largely due to an increase in cell content of a peridinin-chlorophyll a-protein (PCP) and an unidentified chlorophyll a component of the chloroplast membrane. Photosynthetic action spectrum measurements indicated that chlorophyll a, peridinin, and very likely chlorophyll c, were effective light-harvesting pigments for photosynthesis in both high and low light grown cultures of Glenodinium sp. Comparison of action spectra and absorption spectra suggested that low light grown cells selectively increased cellular absorption in the 480 nm to 560 nm region, and effectively utilized this spectral region for the promotion of oxygen evolution.

Purification and characterization of peridinin-chlorophyll a-proteins from the marine dinoflagellates Glenodinium sp. and Gonyaulax polyedra.

A peridinin-chlorophyll a-protein complex (PCP) was obtained in large quantity from the marine dinoflagellates, Glenodinium sp. and Gonyaulax polyedra. The chromoproteins have similar molecular weights, 35,500 for Glenodinium sp. and 34,500 for G. polyedra. The proteins from the PCP complex of Glenodinium sp. dissociated from the chromophore on treatment with 1% sodium dodecyl sulfate (SDS) at room temperature. The protein component was a single subunit with a molecular weight of 15,500. Proteins from the PCP complex of G. polyedra were composed of a single polypeptide with a molecular weight of about 32,000. Two peridinin-chlorophyll a-proteins from Glenodinium sp. accounted for 70% of the PCP complex and had isoelectric points of 7.4 and 7.3. The PCP complex from G. polyedra was dominated by a single chromoprotein with an isoelectric point of 7.2 Chromophore analysis indicated the presence of only peridinin and chlorophyll a in a molar ratio approaching 4:1. Other pigments characteristically found in dinoflagellates were absent. Fluorescence excitation spectra of purified PCP indicated an efficient energy transfer from peridinin to chlorophyll a, an observation that lends support to the reported role of peridinin as an accessory pigment in photosynthetic oxygen evolution. In several other brown colored dinoflagellates examined, PCP representtd less than 20% of the total peridinin. However, no PCP could be isolated from cultures of Amphidinium carterae (PY-1). This study provides further evidence that PCP is a normal component of most peridinin-containing dinoflagellates, and functions as a light-harvesting component of the dinoflagellate chloroplast. No fucoxanthin-containing analog of PCP was detected in the chrysophyte, Cricosphera carterae and the dinoflagellate Glenodinium foliaceum.