Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean

The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.     

Invariable biomass-specific primary production of taxonomically discrete picoeukaryote groups across the Atlantic Ocean

Oceanic photosynthetic picoeukaryotes (< 3 μm) are responsible for > 40% of total primary production at low latitudes such as the North-Eastern tropical Atlantic. In the world ocean, warmed by climate changes, the expected gradual shift towards smaller primary producers could render the role of photosynthetic picoeukaryotes even more important than they are today. Little is still known, however, about how the taxonomic composition of this highly diverse group affects primary production at the basin scale. Here, we combined flow cytometric cell sorting, NaH1?CO? radiotracer incubations and class-specific fluorescence in situ hybridization (FISH) probes to determine cell- and biomass-specific inorganic carbon fixation rates and taxonomic composition of two major photosynthetic picoeukaryote groups on a ～7500-km-long latitudinal transect across the Atlantic Ocean (Atlantic Meridional Transect, AMT19). We show that even though larger cells have, on average, cell-specific CO? uptake rates ～5 times higher than the smaller ones, the average biomass-specific uptake is statistically similar for both groups. On the other hand, even at a high taxonomic level, i.e. class, the contributions to both groups by Prymnesiophyceae, Chrysophyceae and Pelagophyceae are significantly different (P < 0.001 in all cases). We therefore conclude that these group's carbon fixation rates are independent of the taxonomic composition of photosynthetic picoeukaryotes across the Atlantic Ocean. Because the above applies across different oceanic regions the diversity changes seem to be a secondary factor determining primary production.     

A flow cytometric approach to assessing the environmental and physiological status of phytoplankton

Individual particle analysis using a flow cytometer (FCM) was made on natural phytoplankton communities in oligotrophic waters. Our objective was to develop an assay to yield information on the nutrient history of individual cells using FCM. Results from nutrient assays showed that both biovolume and total red fluorescence are affected by the nutrient conditions in the incubator. The light effect was measured by changes in the chlorophyll content of the cells, and after the 12 h incubation the cells seemed well adapted to the light conditions. The estimated kinetic constant for the chlorophyll synthesis averaged 1.5 x 1.0(-2) h-1, whereas the growth rate calculated from the changes in the cell numbers changed from 0.14 to greater than 2.5 doubling per day. The smallest size fraction presented the highest growth rate (greater than 2.5 doublings per day). The relationship between the total red fluorescence as estimated with the FCM and the biovolume revealed that the cells from the 2 m samples at the beginning of the experiment were probably nutrient limited. Important changes in the size of the cells under nutrient limitation were also observed. The FCM data suggest that the FCM is a valuable tool for estimating the relative growth response and nutritional state of natural phytoplankton populations.     

VARIATION IN DCMU-ENHANCED FLUORESCENCE RELATIVE TO CHLOROPHYLL A: CORRELATION WITH THE BROWN TIDE BLOOM1

DCMU–enhanced fluorescence and extracted chlorophyll a were simultaneously measured in Narragansett Bay, Rhode Island and the MERL (Marine Ecosystems Research Laboratory) mesocosms during the 1985 brown tide bloom. Marked differences in the relationship between these variables were observed as the phytoplankton community shifted from dominance by picoalgae to diatoms. The fluorescence to chlorophyll a ratio was significantly (P< 0.05) higher in the mesocosms and the bay when the brown tide species (Aureococcus anophagefferens Hargraves et Sieburth) dominated the phytoplankton community compared with other taxa. Although several factors could have affected the relationship we believe the high ratios are related to the pigment composition and/or small size of the brown tide organism.     

Quantitative assessment of picoeukaryotes in the natural environment by using taxon-specific oligonucleotide probes in association with tyramide signal amplification-fluorescence in situ hybridization and flow cytometry

Picoeukaryotes (cells of <3 micro m in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3- micro m-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.     

Influence of low temperatures on the growth and photosynthetic activity of industrial chicory, <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. partim

The cold stress effect on early vigour and photosynthesis efficiency was evaluated for five industrial chicory varieties with contrasting early vigour. The relationships between the growth and physiological parameters were assessed. The varieties were examined at three growth temperatures: 16 (reference), 8 (intermediate) and 4 °C (stress). The effect was measured using physiological processes (growth, photosynthesis, chlorophyll a fluorescence), and pigment content. The analysis of the measured growth parameters (dry leaf and root mass, and leaf area) indicated that temperature had a significant effect on the varieties, but the overall reaction of the varieties was similar with lowering temperatures. The photosynthesis and chlorophyll a fluorescence measurements revealed significant changes for the photosynthesis (maximum net photosynthesis, quantum efficiency, light compensation point and dark respiration) and chlorophyll a fluorescence parameters (photochemical and non-photochemical quenching) with lowering temperatures for Hera and Eva, two extremes in youth growth. No significant differences could be found between the extremes for the different temperatures. The pigment content analysis revealed significant differences at 4 °C in contrast to 16 and 8 °C, especially for the xanthophyll/carotenoid pool, suggesting a protective role. Subsequently, the relationship between the physiological processes was evaluated using principal component analysis. At 4 °C, 2 principal components were detected with high discriminating power for the varieties and similar classification of the varieties as determined in the growth analysis. This provides a preview on the possible relationships between photosynthesis and growth for industrial chicory at low temperatures.     

Photoadaptation in marine phytoplankton : changes in spectral absorption and excitation of chlorophyll a fluorescence

The optical properties of marine phytoplankton were examined by measuring the absorption spectra and fluorescence excitation spectra of chlorophyll a for natural marine particles collected on glass fiber filters. Samples were collected at different depths from stations in temperate waters of the Southern California Bight and in polar waters of the Scotia and Ross Seas. At all stations, phytoplankton fluorescence excitation and absorption spectra changed systematically with depth and vertical stability of the water columns. In samples from deeper waters, both absorption and chlorophyll a fluorescence excitation spectra showed enhancement in the blue-to-green portion of the spectrum (470-560 nm) relative to that at 440 nm. Since similar changes in absorption and excitation were induced by incubating sea water samples at different light intensities, the changes in optical properties can be attributed to photoadaptation of the phytoplankton. The data indicate that in the natural populations studied, shade adaptation caused increases in the concentration of photosynthetic accessory pigments relative to chlorophyll a. These changes in cellular pigment composition were detectable within less than 1 day. Comparisons of absorption spectra with fluorescence excitation spectra indicate an apparent increase in the efficiency of sensitization of chlorophyll a fluorescence in the blue and green spectral regions for low light populations.     

Trade-off between leaf chlorophyll and betacyanins in Suaeda salsa in the Liaohe estuary wetland in northeast China

Aims Pigment composition is an important functional trait that can be affected by environmental factors. The objective of this study was to investigate the effect of soil salinity on pigment composition in Suaeda salsa by comparing chlorophyll and betacyanin content in the Liaohe estuary wetland, a typical coastal wetland in northeast China.Methods We investigated the plant biomass, percentage of red leaves and pigment content (chlorophyll a, chlorophyll b and betacyanins) in S. salsa in intertidal and supratidal zones of the upper, middle and lower reaches of the Liaohe estuary wetlands. The Na + content of both the soil and plant was also measured. Full analysis of variance and multivariate analysis were used to compare differences in pigment content and Na + content between the supratidal and intertidal zones.Important findings Pigment composition was significantly affected by soil salinity. With increasing soil salinity, the percentage of red leaves was higher in the intertidal zone than in the supratidal zone. In all three reaches, plants had lower chlorophyll a content and higher betacyanin content in the intertidal zone than in the supratidal zone. Compared to chlorophyll a, chlorophyll b was less sensitive to soil salinity. There were no differences in chlorophyll b content between the intertidal and supratidal zones in the upper and lower reaches. Furthermore, pigment composition was associated with both the plant tissue and soil Na + content. Compared to the supratidal zone, the intertidal zone had a higher Na + content in plants. There was a negative relationship between plant chlorophyll content and soil Na + content, but a positive relationship between betacyanin content and soil Na + content. Overall, the results indicated that there might be a trade-off between leaf chlorophyll and betacyanin content in S. salsa to maintain its growth and survival in high salinity environments.     

Physiological responses of estuarine phytoplankton to ultraviolet light-induced fluoranthene toxicity

Polycyclic aromatic hydrocarbons (PAHs) are common pollutants associated with urbanization and suburbanization in estuarine systems, but little is known about their effects on the physiological properties of microalgae. We examined the effects of ultraviolet (UV)-activated fluoranthene toxicity on (a) the growth, chlorophyll a content cell⁻¹, and pigment composition of axenic Ankistrodesmus sp. (an estuarine benthic green microalga) and (b) the phytoplankton population growth and pigment composition of natural communities from an urbanized (Murrells Inlet) vs. forested (North Inlet) salt marsh estuary. The zeaxanthin/violaxanthin ratio increased in Ankistrodesmus sp. cultures exposed to UV light in the presence of fluoranthene, supporting the hypothesis that xanthophyll cycling is an energy dissipative response to photoinduced PAH toxicity in this species. Exposure of natural communities to the combination of UV light and fluoranthene resulted in decreased chlorophyll production and increased zeaxanthin violaxanthin⁻¹ in samples from the urbanized estuary (Murrells Inlet), but not North Inlet, suggesting that phytoplankton in the former “fluoranthene-impacted” estuary were more susceptible to fluoranthene toxicity. Consideration of xanthophyll cycling as a microalgal response to UV-activated PAH toxicity has implications to understanding the influence of these contaminants on microbial food web structure and ecosystem production.     

Differential sunlight sensitivity of picophytoplankton from surface Mediterranean Coastal Waters

We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in Prochlorococcus spp. but not in Synechococcus, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.     

Evolutionary ecology of plant-microbe interactions: soil microbial structure alters selection on plant traits

? Below-ground microbial communities influence plant diversity, plant productivity, and plant community composition. Given these strong ecological effects, are interactions with below-ground microbes also important for understanding natural selection on plant traits? ? Here, we manipulated below-ground microbial communities and the soil moisture environment on replicated populations of Brassica rapa to examine how microbial community structure influences selection on plant traits and mediates plant responses to abiotic environmental stress. ? In soils with experimentally simplified microbial communities, plants were smaller, had reduced chlorophyll content, produced fewer flowers, and were less fecund when compared with plant populations grown in association with more complex soil microbial communities. Selection on plant growth and phenological traits also was stronger when plants were grown in simplified, less diverse soil microbial communities, and these effects typically were consistent across soil moisture treatments. ? Our results suggest that microbial community structure affects patterns of natural selection on plant traits. Thus, the below-ground microbial community can influence evolutionary processes, just as recent studies have demonstrated that microbial diversity can influence plant community and ecosystem processes.     

Chlorophyll precursors in the plasma membrane of a cyanobacterium, Anacystis nidulans. Characterization of protochlorophyllide and chlorophyllide by spectrophotometry, spectrofluorimetry, solvent partition, and high performance liquid chromatography

Plasma membranes were isolated and separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation of crude membranes prepared by French pressure cell extrusion of lysozyme-treated Anacystis nidulans. Two distinct populations of chlorophyll-free plasma membrane vesicles were obtained exhibiting buoyant densities of 1.087 and 1.100 g/cm3 as opposed to a uniform density of 1.192 g/cm3 for thylakoid membranes. Plasma and thylakoid membranes were characteristically different also with respect to fatty acid and protein composition, cytochrome oxidase activity, and pigment content as analyzed by spectrophotometry, spectrofluorimetry, and high performance liquid chromatography. Apart from carotenoids, chlorophyll a was the only major photosynthetic pigment detected in thylakoid membranes while plasma membranes contained virtually no chlorophyll a but (besides large amounts of carotenoids) protochlorophyllide a and chlorophyllide a as revealed by solvent partition (between n-hexane and acetone or methanol), room and low temperature fluorescence emission and excitation spectra, and analytical separation and identification by high performance liquid chromatography and comparison with authentic standards. The protochlorophyllide in the plasma membrane could be transformed into chlorophyllide in the dark in vitro by incubating the membrane preparation with NADPH; NADP+ effected the reverse transition.     

Delayed luminescence of Prorocentrum minimum under controlled conditions

Delayed luminescence (DL), also termed delayed fluorescence or delayed light emission, is the phenomenon of long-lived light emission by plants and cyanobacteria after being illuminated with light and put into darkness. Culture growth of three Prorocentrum minimum strains was studied with DL measurements. DL decay kinetics was measured from 1–60 s after a pulse of white light. The strains used were from the Adriatic Sea (PmK), from Chesapeake Bay, USA (D5), and from the Baltic Sea (BAL), cultured at salinity of 32, 16, and 8 (practical salinity scale), respectively. The strains differed in cell size and chlorophyll a content (PmK > D5 > BAL), as well as in DL parameters. The DL results were compared to standard measurements of culture density and carbon content (calculated from biovolumes). DL decay curves had a specific peak, which changed with culture growth and showed more similarities between the strains PmK and D5. The DL intensity increased with cell density and carbon content in a two-stage process, corresponding to the lag and exponential phases of growth. DL intensity was best correlated with carbon content irrespective of strain and is proposed as an estimate of biomass and for differentiating between lag and exponential phases of growth.     

Differential Sunlight Sensitivity of Picophytoplankton from Surface Mediterranean Coastal Waters

We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in Prochlorococcus spp. but not in Synechococcus, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.     

Interactions of lichens with heavy metals

Recent developments in knowledge about the interactions between lichens and heavy metals at different levels, from populations to cells and from ecology to molecular biology are reviewed. Sources of heavy metals, mechanisms of heavy metal accumulation and detoxification by lichens are discussed. Special emphasis is placed on ultrastructural changes as well as physiological parameters such as membrane integrity, pigment composition, chlorophyll a fluorescence, photosynthesis, respiration, contents of ATP, amino acids, ergosterol, ethylene, non-protein thiols, activity of antioxidant enzymes and expression of stress proteins.     

内陆湖泊主要藻种散射特性

通过室内培养4种主要淡水藻种--铜绿微囊藻(Microcystis aeruginosa)、普通小球藻(Chlorella vulgaris)、梅尼小环藻(Cyclotella meneghiniana)以及卵形隐藻(Cryptomonas ovata), 测定其散射和后向散射特征参数、用单位叶绿素a浓度的散射值和后向散射值来表征4种藻的散射和后向散射能力。结果表明, 铜绿微囊藻散射和后向散射能力最强, 其次为梅尼小环藻, 普通小球藻的能力最弱。通过计算后向散射概率, 显示铜绿微囊藻和梅尼小环藻的后向散射概率值较高, 普通小球藻和卵形隐藻的后向散射概率值较低。后向散射特性影响因子分析显示, 影响后向散射值的主要因素有叶绿素a浓度及藻蓝蛋白色素比例。当叶绿素a浓度不断增加时, 其后向散射值不断增大; 当藻类所含叶绿素a比重不断上升时, 其后向散射值也不断增大。而细胞粒径与后向散射值之间未表现出很好的相关性。因此, 通过单位叶绿素a散射和后向散射概率特征可以辨别出藻细胞形态较为接近的铜绿微囊藻和普通小球藻。     

Effect of anthocyanins, carotenoids, and flavonols on chlorophyll fluorescence excitation spectra in apple fruit: signature analysis, assessment, modelling, and relevance to photoprotection

Whole apple fruit (Malus domestica Borkh.) widely differing in pigment content and composition has been examined by recording its chlorophyll fluorescence excitation and diffuse reflection spectra in the visible and near UV regions. Spectral bands sensitive to the pigment concentration have been identified, and linear models for non-destructive assessment of anthocyanins, carotenoids, and flavonols via chlorophyll fluorescence measurements are put forward. The adaptation of apple fruit to high light stress involves accumulation of these protective pigments, which absorb solar radiation in broad spectral ranges extending from UV to the green and, in anthocyanin-containing cultivars, to the red regions of the spectrum. In ripening apples the protective effect in the blue region could be attributed to extrathylakoid carotenoids. A simple model, which allows the simulation of chlorophyll fluorescence excitation spectra in the visible range and a quantitative evaluation of competitive absorption by anthocyanins, carotenoids, and flavonols, is described. Evidence is presented to support the view that anthocyanins, carotenoids, and flavonols play, in fruit with low-to-moderate pigment content, the role of internal traps (insofar as they compete with chlorophylls for the absorption of incident light in specific spectral bands), affecting thereby the shape of the chlorophyll fluorescence excitation spectrum.     

Effect of light quality on the growth and proximal composition of <Emphasis Type="Italic">Amphora</Emphasis> sp.

We determined the effects of various light spectra (white, blue, green, yellow, and Grolux) on the growth rate, proximate composition, pigment content, and cell size of the marine benthic diatom Amphora sp. during two growth phases of batch cultures. The growth rate was higher under green light and lowest with Grolux and yellow light. Cell size differed significantly between growth phases but remained unaffected by light spectra; the smallest cells were observed on the initial day of culture, whereas larger cells developed in the stationary phase under Grolux treatment. The proximate composition was modified by the light spectra and growth phase. In the exponential growth phase, the protein content was higher with yellow and white light, and lipid content peaked with Grolux. The pigment content (chlorophyll a, carotenoids) was also higher under yellow light. In the stationary growth phase, we noted a higher carbohydrate content under Grolux and yellow light. Our results show that light spectra and growth phase influence the metabolism of Amphora sp., changing its proximate composition, pigment content, growth rate, and cell size.     

Composition of the summer photosynthetic pico and nanoplankton communities in the Beaufort Sea assessed by T-RFLP and sequences of the 18S rRNA gene from flow cytometry sorted samples

The composition of photosynthetic pico and nanoeukaryotes was investigated in the North East Pacific and the Arctic Ocean with special emphasis on the Beaufort Sea during the MALINA cruise in summer 2009. Photosynthetic populations were sorted using flow cytometry based on their size and pigment fluorescence. Diversity of the sorted photosynthetic eukaryotes was determined using terminal-restriction fragment length polymorphism analysis and cloning/sequencing of the 18S ribosomal RNA gene. Picoplankton was dominated by Mamiellophyceae, a class of small green algae previously included in the prasinophytes: in the North East Pacific, the contribution of an Arctic Micromonas ecotype increased steadily northward becoming the only taxon occurring at most stations throughout the Beaufort Sea. In contrast, nanoplankton was more diverse: North Pacific stations were dominated by Pseudo-nitzschia sp. whereas those in the Beaufort Sea were dominated by two distinct Chaetoceros species as well as by Chrysophyceae, Pelagophyceae and Chrysochromulina spp.. This study confirms the importance of Arctic Micromonas within picoplankton throughout the Beaufort Sea and demonstrates that the photosynthetic picoeukaryote community in the Arctic is much less diverse than at lower latitudes. Moreover, in contrast to what occurs in warmer waters, most of the key pico- and nanoplankton species found in the Beaufort Sea could be successfully established in culture.     

Acclimation of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> to the light environment: the relationship between photosynthetic function and chloroplast composition

Plants respond to growth under different environmental conditions by adjusting the composition of the photosynthetic apparatus. To investigate the consequences of the acclimation strategies adopted by Arabidopsis thaliana, we have assessed the functioning of the photosynthetic apparatus in plants with very different chloroplast compositions. Using chlorophyll fluorescence analysis, we have determined the efficiency of, and capacity for, electron transport, assessed the ability to undergo state transitions, and measured non-photochemical quenching over a range of actinic irradiances followed by its resolution into fast- and slow-relaxing components; parallel measurements of leaf carotenoid composition were also carried out. The data clearly show that acclimation serves to maintain the electron transport chain in an oxidised state, ensuring efficient photochemistry. Furthermore, plants grown in high light have a greater capacity for energy-dependent feedback de-excitation, but this is not correlated with xanthophyll cycle pigment levels or de-epoxidation state. Surprisingly, even plants with very low levels of light-harvesting complexes were able to undergo state transitions. We also show that apparent discrepancies between chloroplast composition and photosynthetic function can be attributed to varying degrees of light penetration through the leaf. Thus, leaf chlorophyll content is an important factor influencing acclimation within the leaf.