Chlorophyll C Research Articles

Biosynthesis of chlorophyll c in a dinoflagellate and heterologous production in planta

Chlorophyll c is a key photosynthetic pigment that has been used historically to classify eukaryotic algae. Despite its importance in global photosynthetic productivity, the pathway for its biosynthesis has remained elusive. Here we define the CHLOROPHYLL C SYNTHASE (CHLCS) discovered through investigation of a dinoflagellate mutant deficient in chlorophyll c. CHLCSs are proteins with chlorophyll a/b binding and 2-oxoglutarate-Fe(II) dioxygenase (2OGD) domains found in peridinin-containing dinoflagellates; other chlorophyll c-containing algae utilize enzymes with only the 2OGD domain or an unknown synthase to produce chlorophyll c. 2OGD-containing synthases across dinoflagellate, diatom, cryptophyte, and haptophyte lineages form a monophyletic group, 8 members of which were also shown to produce chlorophyll c. Chlorophyll c1 to c2 ratios in marine algae are dictated in part by chlorophyll c synthases. CHLCS heterologously expressed in planta results in the accumulation of chlorophyll c1 and c2, demonstrating a path to augment plant pigment composition with algal counterparts.

Light intensity changes and UVB radiation affect peridinin content and antioxidant activity in the Cassiopea andromeda holobiont

IntroductionThe up-side down jellyfish Cassiopea andromeda represents a yet untapped marine species that could be targeted as a new source for functional ingredients, such as natural pigments and antioxidants. Since C. andromeda hosts endosymbiotic dinoflagellates, this jellyfish contains peridinin pigments, which are linked with high antioxidant capacities and many other health-promoting properties. This study investigates the potential to specifically increase the content of peridinin and overall antioxidant activity in C. andromeda, through the targeted application of different photosynthetic active radiation (PAR) intensities and ultraviolet radiation, cultured in fully controlled indoor aquaculture systems.Materials and MethodsIndoor bred C. andromeda specimens were exposed to five different PAR intensities (50, 100, 200, 400 and 800 µmol photons m−2 s−1) and a combined treatment of narrow-band UVB (λ = 285 ± 10 nm) radiation and intermediate (200 µmol photons m−2 s−1) PAR intensity over a period of four weeks. Before the treatment and after two- and four-week treatment intervals, pigment concentrations and antioxidant activity levels were measured using high-performance liquid chromatography (HPLC) and the Trolox Equivalent AntioxidantCapacity (TEAC) assay, respectively. In addition, relative growth rate, umbrella pulsation and photosynthetic efficiency (measured by pulse amplitude modulated fluorometry) of C. andromeda individuals were also monitored throughout the experiment. ResultsChlorophyll a (Chl a) and peridinin (Per) dominated overall pigment content in C. andromeda endosymbionts, chlorophyll c2 and diadinoxanthin were detected in minor amounts. Over the treatment time, Chl a and Per concentrations, measured as µg g−1 jellyfish dry weight and pg microalgae-cell−1, decreased sharply at higher PAR intensities (200 – 800 µmol photons m−2 s−1) compared to the control treatment (100 µmol photons m−2 s−1). After four weeks Chl a and Per concentrations were lowest at the highest PAR intensity (800 µmol photons m−2 s−1) and highest at the lowest PAR intensity (50 µmol photons m−2 s−1). Moreover, the ratio of Chl a and Per showed a relative decrease of Per with increasing PAR intensity. The combined treatment of narrow-band UVB (λ = 285 ± 10 nm) radiation and intermediate (200 µmol photons m−2 s−1) PAR intensity led to significantly elevated Chl a and Per concentrations compared to the 200 µmol photons m−2 s−1 PAR treatment without UVB. Significantly elevated antioxidant activity levels, measured as Trolox Equivalents mmol g−1 jellyfish dry weight, were only detected in UVB exposed C. andromeda, indicating that Chl a and Per did not determine overall antioxidant capacity. The photosynthetic efficiency of C. andromeda endosymbionts was not affected by elevated antioxidant activity. Opposing that, the jellyfish hosts that were exposed to the UVB treatment shrunk drastically, indicating a strong stress response.DiscussionWith this study, we demonstrate for the first time the application potential of PAR intensity manipulations and UVB irradiation, to increase the content of valuable pigments and antioxidants in C. andromeda jellyfish and their endosymbiotic dinoflagellates that live in hospite within the host tissue. Based on these findings, we propose the culture of C. andromeda under fully controlled and light-optimized conditions as new pathway to harness bioproducts and functional ingredients.

Morphological and molecular comparison as a useful tool for identification of the three centric marine diatoms (Bacillariophyceae: Chaetoceros).

The objective of this study was to identify morphological and molecular comparison of three marine Chaetoceros species using microscopic observations, sequence analysis of 18S rDNA, random amplified polymorphic DNA (RAPD-PCR) barcoding and nuclear magnetic resonance (NMR) spectroscopy.Chaetoceros were obtained from three different algae laboratories: Center of Excellence for Marine Biotechnology (CEMB), Chanthaburi Coastal Fisheries Research and Development (CHAN) and Institute of Marine Science, Burapha University (BIM). Genomic DNA for the RAPD-PCR analysis was extracted using the phenol-chloroform method, followed by 18S rDNA amplification.The blast results of 18S rDNA sequence confirmed the significantly matched to C. gracilis for Chaetoceros BIM and CHAN and C. muelleri for Chaetoceros CEMB(e-value = 0.0, identity = 99%). The RAPD-PCR results revealed differences in the three Chaetoceros isolates with polymorphisms between 30.43% and 60.00%, and Chaetoceros CEMB showed high polymorphic bands. Scanning electron microscopy revealed that Chaetoceros CEMB were larger and had larger setae compared to the other isolates (P < 0.05). The results of the NMR characterization of metabolites were consistent with the results of the sequence and morphological analyses. The concentrations of several metabolites, including chlorophyll c1, chlorophyll a, Myo-inositol, fucoxanthin, astaxanthin, lutein and zeaxanthin, were lower in Chaetoceros CEMB than in Chaetoceros BIM and CHAN. However, high concentrations of fatty acids, such as oleic acid, linoleic acid, α-linolenic acid and arachidic acid, were observed in all isolates. Generally, the results of this study will aid future studies examining the diversity of Chaetoceros in various cultural environments.

Hydrobiological and Bio-Optical Characterization of a Red Tide Occurredin Meda Creek, Porbandar, India

Red tides are becoming more common in aquatic ecosystems, particularly the coastal habitats which are highly influenced by climatic and anthropogenic activities. We provide the first report of a red tide event in Meda creek, west coast of India. Light and scanning electron microscopy were used to identify the bloom forming organism, while physicochemical, biological and biooptical parameters were determined to characterize the aquatic body of the Meda Creek. The dominant bloom forming organisms were observed as unarmored, spherical, dorsoventrally compressed flagellate with truncated apex, notched and rounded hypocone, displaced premedian cingulum and extending sulcus. The length wise size of the cell varied from 20 to 30.83μM, whereas, the width varied from 14 to 27.5μM. The organisms was identified as Gymnodinium sp. High Performance Liquid Chromatography (HPLC) and spectral profile of the bloom indicatedeleven major peaks corresponding to that of 19-but-fucoxanthin (52.75%), astaxanthin (9.62%), fucoxanthin (8.81%), Mgdv (6.45%), prasinoxanthin (3.35%), chlorophyll C2 (3.22%), neoxanthin (3.12%), antheraxanthin (3.14%), diadinoxanthin (2.63%) and diatoxanthin (1.38%). Chlorophyll a concentration in the bloom sample was 38.8μg/L. Water temperature, salinity, NH3-N, NO2-N, NO3-N, PO4-Pwere higher during the bloom period than the non-blooming ones, while metals including Fe, Zn, Cr, Mn and Co were lower. No death of aquatic biota was observed. We propose that eutrophication along with the warm and saline conditions provided enabling conditions for occurrence the bloom. Also, most of the pigments expressed have antioxidant role and might be purposeful for the survival of the organism in the extreme condition of temperature and salinity recorded. Proper management and monitoring measures might help mitigate further occurrences of this bloom in the light changing climate and increasing anthropogenic impacts.

Apparent shifts in the microplankton community in response to zinc and iron enrichments in the Northeastern Subarctic Pacific

The offshore Gulf of Alaska—in the northeastern subarctic Pacific—is a high-nitrate, low-chlorophyll (HNLC) oceanic region where concentrations of dissolved iron (DFe) and zinc (DZn) are of the order ~0.1 nmol kg−1 in surface waters. At the average winter mixed layer depth of ~120 m, DFe and DZn are of the orders ~0.1–0.2 nmol kg−1 and ~1–2 nmol kg−1, respectively. Vertical supply of Fe to surface waters is therefore limited, with phytoplankton blooms driven by episodic atmospheric and lateral inputs of Fe. In contrast, DZn is presumably replenished through seasonal vertical mixing, though maintained at low levels in surface waters, even in winter, implying that removal processes are in operation. Previous supplementation experiments have demonstrated that phytoplankton biomass (chl-a), growth, and drawdown of nitrate (NO3-) are stimulated strongly by Fe but only minimally, if at all, by Zn alone. Using on-deck bioassay incubations of surface waters, we confirm that the addition of Fe stimulates significant increases in chl-a and large diatoms, and drawdown of NO3- and silicic acid [Si(OH)4]. Associated drawdown of DZn and dissolved cadmium (DCd) indicated Zn stress in the control and +Fe treatment. Supplementation with Zn alone had no significant impact on NO3- and commonly monitored pigments such as chl-a and fucoxanthin. However, in the +Zn treatment, DFe and coccolithophore abundance were significantly lower, and the concentration of particulate organic carbon (POC) and that of the pigments alloxanthin and chlorophyll c1+2 were significantly higher than those in control incubations. Our experiment corroborates previously observed relationships between DZn and alloxanthin and chlorophyll c across the subarctic north Pacific and Bering Sea and could help to explain the low or undetectable concentrations of alloxanthin frequently observed in this region. Alloxanthin is a pigment specific to the cryptophytes, either within free-living cells or within functional plastids retained by mixotrophic protists. Both cryptophytes and mixotrophic ciliates, such as Laboea sp. and Strombidium spp., make a variable but significant contribution to phytoplankton biomass in this area. In the absence of Fe supply to surface waters, Zn could play a hitherto unsuspected role in shaping plankton communities in the HNLC Gulf of Alaska.

Pigment-based chemotaxonomy of the phytoplankton community from a marine mussel farming area

Microscopy technique is traditionally employed for assessing phytoplankton taxonomy. However, chemical approaches, such as the identification of biomarker pigments, allow to elucidate microalgae groups more quickly, in addition to detecting these valuable compounds. This study aimed to identify the pigment profile of microalgae species and to determine the seasonal pigment fluctuation in a marine mussel farm. Water samples were collected periodically between the years of 2014 and 2015. Phytoplanktonic organisms were isolated and cultivated in f/2 liquid medium under controlled temperature and light cycle. Cell content extracts from each unialgal culture and water sample were analyzed by High Performance Liquid Chromatography (HPLC). Pigment profiles of 13 marine microalgae strains were identified (12 Diatoms and 1 Dinoflagellate). Chlorophyll a and fucoxanthin were most present in Diatoms, while peridinin was the major carotenoid in Prorocentrum micans. Bacillariophyceae had higher density in the summer and fall. Dinophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, Dictyophyceae and Ebriidea, had an increase in density during winter and spring. Following the trend of phytoplankton groups, pigments such as fucoxanthin, zeaxanthin, diadinoxanthin, chlorophyll c3 and c2, were identified throughout the year. Prasinoxanthin, peridinin, dinoxanthin and antheraxanthin were identified in samples collected during winter and spring.

Comparative analyses of nutritional strategies among the species within the genus Paragymnodinium (Gymnodiniales, Dinophyceae).

The genus Paragymnodinium exhibits various nutritional strategies despite the fact that each species has pigmented plastids. This provides an excellent opportunity to investigate the evolution of the mode of nutrition and the function of the plastids through comparative studies. In this study, we analyzed the growth, pigment composition, absorption spectra, variable chlorophyll a fluorescence, and photosynthetic carbon fixation capabilities of P. stigmaticum, P. asymmetricum, and P. inerme. The autotrophic species P. asymmetricum and P. inerme without resorting to any nutrition from prey organisms displayed high PSII activity and carbon fixation rates. The pigment compositions of these two species were identical to those of other typical peridinin-containing type dinoflagellates. On the contrary, the phagotrophic species P. stigmaticum showed heterotrophic growth, and the variable chlorophyll a fluorescence properties and carbon fixation rates indicated significantly lower photosynthetic competence relative to those of the above two species. Paragymnodinium stigmaticum also contained peridinin, but pigment content ratios of peridinin, chlorophyll c2, and β-carotene were significantly different from those of other two species. The absorption spectrum analysis revealed a redshift in the peak of the Qy band of chlorophyll a in P. stigmaticum, presumably due to a change in chlorophyll-protein complexes. We conclude that such distinct differences in nutritional strategies between members of the genus Paragymnodinium would provide a platform for the hypothetical loss of photosynthetic function leading to colorless dinoflagellates.

Living Inside a Jellyfish: The Symbiosis Case Study of Host-Specialized Dinoflagellates, “Zooxanthellae”, and the Scyphozoan Cotylorhiza tuberculata

The photosymbiosis with host-specific dinoflagellates is a widespread relationship in marine organisms. Despite the evidenced biodiversity of this kind of mutualism, most research focuses on the study of scleractinian corals, and there is a lack of knowledge about other symbiotic cnidarians such as jellyfishes. The Mediterranean jellyfish Cotylorhiza tuberculata (Rhizostomae, Scyphozoa) harbors an endosymbiotic dinoflagellate of the family Symbiodiniaceae. In this study, we examine the algae distribution within the host body as well as, the pigment content and cell density of the symbiont. Furthermore, the size, morphology and fluorescence of cultured symbionts were studied under light microscopy, Imaging Flow Cytometry (IFC), and Scanning Electron Microscopy (SEM). The C:N composition and optical properties of the medusa tissue were measured to evaluate their role in the symbiosis. The medusae body was divided into two different sections to investigate the distribution of symbionts in hospite: oral arms (OA) and umbrella (UM). C:N composition of C. tuberculata was and symbiont density was significantly higher in the OA section. Mean chlorophyll a concentration of the algae was 1.33 (± 0.83) pg Chl a cell–1. The study of the pigment composition by HPLC (High Performance Liquid Chromatography), revealed the presence of 13 different pigments, being the most representative chlorophyll a, chlorophyll c2, and peridinin typical pigments of Symbiodiniaceae. Cell diameter of algae freshly isolated from the host was 8.71 ± 0.97 μm and cell growth rate was 0.52 (± 0.09) 106 cell ml–1 d–1. The presence of vegetative coccoid cells, doublet and motile mastigotes were revealed within the Symbiodiniaceae cultures. A calcifying matrix typical of Symbiodiniaceae and formed in partner with bacteria, was also observed most cultures. The umbrella tissue of the medusa absorbed at ultraviolet radiation (UVR) region, suggesting that medusae tissue protects photosymbionts from the negative effect of the high energetic UVR and attenuates the light intensity reaching algae inside the host. The presence of a dense Symbiodiniaceae population and the protection to UVR and elevated environmental irradiance provided by medusae tissue, maintain symbionts in optimal light conditions for photosynthesis and may be a reason added to explain the population success of Cotylorhiza tuberculata.

Optical Detection of Harmful Algal Blooms in the Belgian Coastal Zone: A Cautionary Tale of Chlorophyll c3

Phaeocystis globosa is a nuisance haptophyte species that forms annual blooms in the southern North Sea and other coastal waters. At high biomass concentration, these are considered harmful algal blooms due to their deleterious impact on the local ecosystems and economy, and are considered an indicator for eutrophication. In the last two decades, methods have been developed for the optical detection and quantification of these blooms, with potential applications for autonomous in situ or remote observations. However, recent experimental evidence suggests that the interpretation of the optical signal and its exclusive association with P. globosa may not be accurate. In the North Sea, blooms of P. globosa are synchronous with those of the diatom Pseudo-nitzschia delicatissima, another harmful bloom-forming species with similar pigmentation and optical signature. Here we combine new and published measurements of pigmentation composition and inherent optical properties from pure cultures of several algal and cyanobacterial groups, together with environmental spectroscopy data, to identify the pigments generating the optical signals captured by two established algorithms. We further evaluate the association of those pigments and optical signals with P. globosa. We found that the interpretation of the pigment(s) generating the optical signals were incorrect and that previous methods are not specific to P. globosa, even in the context of the phytoplankton assemblage of the southern North Sea. Additionally, we found that the optical and pigment signatures of Phaeocystis species are part of a broad pigmentation trend across unrelated taxonomic groups related to chlorophyll c3 presence, with important consequences for the interpretation of pigment and optical data. We then develop and evaluate an algorithm to detect this pigmentation pattern with minimal influence of co-occurring species and elaborate general recommendations for the future development of algorithms.

Its What’s on the Inside That Counts: An Effective, Efficient, and Streamlined Method for Quantification of Octocoral Symbiodiniaceae and Chlorophyll

Ocean warming driven bleaching is one of the greatest threats to zooxanthellate cnidarians in the Anthropocene. Bleaching is the loss of Symbiodiniaceae, chlorophyll, or both from zooxanthellate animals. To quantify bleaching and recovery, standardised methods for quantification of Symbiodiniaceae and chlorophyll concentrations have been developed for reef-building scleractinian corals, but no such standard method has been developed for octocorals. For stony corals, quantification of Symbiodiniaceae and chlorophyll concentrations often relies on normalisation to skeletal surface area or unit of biomass [i.e., protein, ash-free dry weight (AFDW)]. Stiff octocorals do not change their volume, as such studies have used volume and surface area to standardise densities, but soft-bodied octocorals can alter their size using water movement within the animal; therefore, Symbiodiniaceae and chlorophyll cannot accurately be measured per unit of surface area and are instead measured in units of Symbiodiniaceae and chlorophyll per μg of host protein or AFDW. Though AFDW is more representative of the full biomass composition than host protein, AFDW is more time and resource intensive. Here, we provide a streamlined methodology to quantify Symbiodiniaceae density, chlorophyll concentration, and protein content in soft-bodied octocorals. This technique uses minimal equipment, does not require freeze-drying or burning samples to obtain ash weight, and is effective for down to 0.2 g wet tissue. Bulk samples can be centrifuged, the Symbiodiniaceae pellet washed, and the supernatant saved for protein analysis. This efficient technique allows for clean, easy to count samples of Symbiodiniaceae with minimal animal protein contamination. Chlorophyll a and c2 extractions occurs at different rates, with chlorophyll a taking 24 h to extract completely at 4°C and chlorophyll c2 taking 48 h. Finally, we found that where necessary, wet weight may be used as a proxy for protein content, but the correlation of protein and wet weight varies by species and protein should be used when possible. Overall, we have created a rapid and accurate method for quantification of bleaching markers in octocorals.

Pigment content analysis in two HAB forming dinoflagellate species during the growth period

Dinoflagellates form a large group of microalgae that have different pigments and the pigment content can vary depending on environmental conditions such as temperature, salinity, and their growth phase. They have the potential to cause red tides in coastal waters, so it is important to identify their pigments. In this study, two bloom forming dinoflagellate species, Scrippsiella trochoidea and Gyrodinium instriatum from the Oman Sea, were studied for their pigment content by HPLC every 5 days over a 30-day growth period. In both species, 10 pigment types were identified: unknown carotenoids, chlorophyll (chl) c2, peridinin, peridinin-like, diadinochrome, diadinoxanthin, dinoxanthin, diatoxanthin, chl a, and β,β-carotene. Although both species have the same pigment types, they vary in concentration in different days of growth. The highest cell density in S. trochoidea species was on day 15 of the growth period and the predominant pigments were chl c2, diadinoxanthin, and chl a. Peridinin, peridinin-like, diadinochrome, and β,β-carotene were predominant pigments on day 10, before the cell reached its maximum density. In G. instriatum, the highest cell density during the growth period was in day 10. The highest levels of unknown carotenoids, chl c2, diadinoxanthin, dinoxanthin, peridinin, peridinin-like, diadinochrome, and β,β-carotene pigments were measured on day 25 of growth. The type of chloroplast in both species was the peridinin type. In interpreting the pigment concentration and type, the age of the species should be considered.

Absorption and Fluorescence Spectral Database of Chlorophylls and Analogues.

Absorption spectra and fluorescence spectra are essential for use across the photosciences, yet such spectra along with the all-important values for molar absorption coefficient (ε) and fluorescence quantum yield (Φf ) often are found with great difficulty. Here, a literature survey concerning the vital class of chlorophyll compounds has led to identification of spectra for 150 members. Spectra in print form have been digitized (with baseline corrections) and assembled into a database along with literature references, solvent identity and values for ε and Φf (where available). The database encompasses photosynthetic tetrapyrroles wherein the chromophore is a porphyrin (e.g. chlorophyll c1 , protochlorophyll a), chlorin (e.g. chlorophyll a, bacteriochlorophyll c) or bacteriochlorin (e.g. bacteriochlorophyll a). Altogether, the database contains 305 absorption spectra (from 19 porphyrins, 109 chlorins and 22 bacteriochlorins) and 72 fluorescence spectra (from 10 porphyrins, 30 chlorins and 4 bacteriochlorins). The spectral database should facilitate comparisons and quantitative calculations. All spectra are available in print form in the Supporting Information. The entire database in digital form is available with the PhotochemCAD program for free downloading and further use at http://www.photochemcad.com.

Revealing vibronic coupling in chlorophyll c1 by polarization-controlled 2D electronic spectroscopy

Vibronic coupling between molecules has been recently discussed to play an important role in photosynthetic functions. Furthermore, this type of coupling between electronic states has been suggested to define photophysical properties of chlorophylls, a family of photosynthetic molecules. However, experimental investigation of vibronic coupling presents a major challenge. One subtle way to study vibronic coupling is by excitation and observation of superpositions of vibrational states via transitions to vibronically mixed states. Such superpositions, called coherences, are then observed as quantum beats in non-linear spectroscopy experiments. Here we present polarization-controlled two-dimensional electronic spectroscopy study of the chlorophyll c1 molecule at cryogenic (77 K) temperature. By applying complex analysis to the oscillatory signals we are able to unravel vibronic coupling in this molecule. The vibronic mixing picture that we see is much more complex than was thought before.

Spatial-temporal variation of Aureococcus anophagefferens blooms in relation to environmental factors in the coastal waters of Qinhuangdao, China

The brown tides occurring in the coastal scallop cultivation area of Qinhuangdao, China, in recent years are caused by Aureococcus anophagefferens and significantly impact the scallop industry and the marine ecosystem in this region. Long-term investigations of phytoplankton and hydrological variables in the Qinhuangdao sea area were conducted in this study to understand the spatial-temporal variations of A. anophagefferens in relation to environmental factors. Samples were collected during twelve cruises from July 2011 to December 2013 and were analyzed for the temperature, salinity, dissolved oxygen (DO), nutrients and phytoplankton pigments. All diagnostic pigments of A. anophagefferens, such as chlorophyll c3 (Chl c3), Chl c2, 19′-butanoyloxyfucoxanthin (But-fuco), fucoxanthin (Fuco), and diadinoxanthin (Diad), were detected in the surface water by using high-performance liquid chromatography (HPLC). The highest concentrations of But-fuco (5.64 μg L−1), Fuco (37.94 μg L−1) and chlorophyll a (Chl a, 17.25 μg L−1) occurred in different seasons and sampling sites. The A. anophagefferens bloom (as indicated by But-fuco) usually expanded from the south to the north of the Qinhuangdao sea area, close to scallop-culturing regions. The bloom unusually starts in May, reaches its peak in June and almost disappears in August, with the temperature ranging from ca. 19 °C to 23 °C. The redundancy analysis (RDA) indicated that relatively high salinity (>29) and low inorganic nutrients were suitable for the development of the A. anophagefferens bloom. The ratios of diagnostic pigments to Chl a were not constant during different cruises and generally obeyed two different linear relationships, thus indicating the co-occurrence of the blooms of A. anophagefferens and other species, such as Minutocellus polymorphus. In summary, our work reports the long-term variation of A. anophagefferens blooms based on diagnostic pigments and environmental controls, which may provide more insights into the formation mechanisms of the brown tide in this region.

Identification of candidate genes related to salt tolerance of the secretohalophyte Atriplex canescens by transcriptomic analysis

BackgroundAtriplex canescens is a typical C4 secretohalophyte with salt bladders on the leaves. Accumulating excessive Na+ in tissues and salt bladders, maintaining intracellular K+ homeostasis and increasing leaf organic solutes are crucial for A. canescens survival in harsh saline environments, and enhanced photosynthetic activity and water balance promote its adaptation to salt. However, the molecular basis for these physiological mechanisms is poorly understood. Four-week-old A. canescens seedlings were treated with 100 mM NaCl for 6 and 24 h, and differentially expressed genes in leaves and roots were identified, respectively, with Illumina sequencing.ResultsIn A. canescens treated with 100 mM NaCl, the transcripts of genes encoding transporters/channels for important nutrient elements, which affect growth under salinity, significantly increased, and genes involved in exclusion, uptake and vacuolar compartmentalization of Na+ in leaves might play vital roles in Na+ accumulation in salt bladders. Moreover, NaCl treatment upregulated the transcripts of key genes related to leaf organic osmolytes synthesis, which are conducive to osmotic adjustment. Correspondingly, aquaporin-encoding genes in leaves showed increased transcripts under NaCl treatment, which might facilitate water balance maintenance of A. canescens seedlings in a low water potential condition. Additionally, the transcripts of many genes involved in photosynthetic electron transport and the C4 pathway was rapidly induced, while other genes related to chlorophyll biosynthesis, electron transport and C3 carbon fixation were later upregulated by 100 mM NaCl.ConclusionsWe identified many important candidate genes involved in the primary physiological mechanisms of A. canescens salt tolerance. This study provides excellent gene resources for genetic improvement of salt tolerance of important crops and forages.

Understanding the nitrogen uptake and assimilation of the Chinese strain of Aureococcus anophagefferens (Pelagophyceae)

Brown tides caused by Aureococcus anophagefferens have occurred annually and severely impacted on the local ecology and economy in the coastal waters of Qinhuangdao, China since 2009. The uptake preference, pigment composition and growth responses of the Chinese strain of A. anophagefferens to different nitrogen substrates were studied in the laboratory to better understand its nitrogen (N) strategies and compare the findings with those from the other strains in the U.S.A. Our results indicated that A. anophagefferens had a high absolute uptake rate (ρmax) for NH4+ and low half-saturation constants (Ks) for NO3− and urea. It grew faster on the organic substrates (urea and glutamic acid) than that on the inorganic substrates (NO3− and NH4+). A. anophagefferens had higher contents of cellular pigments for both photosynthesis (chlorophyll a, chlorophyll c2, 19′-butanoyloxyfucoxanthin and fucoxanthin) and photoprotection (β-Carotene and diadinoxanthin) on urea than did on NO3− and NH4+, suggesting a stronger capability for photosynthesis on urea. A. anophagefferens had higher contents of cellular carbon, N and proteins on urea than did on NO3− and NH4+, suggesting a stronger capability for the acclimation and storage of C and N on urea. A. anophagefferens produced more β-carotene rather than chlorophyll a on glutamic acid and thus inhibited photosynthesis and carbon fixation. We found in this study that urea was the best N substrate for the growth of A. anophagefferens. A. anophagefferens might have a competitive advantage over other phytoplankton in waters enriched with DON, particularly with urea.

Evidence of photoacclimatization at mesophotic depths in the coral-Symbiodinium symbiosis at Flower Garden Banks National Marine Sanctuary and McGrail Bank

Similar to shallower conspecifics, mesophotic scleractinian corals found at ~ 30–150 m depths maintain important symbioses with photosynthetic microalgae in the genus Symbiodinium. Despite the importance of coral-algal symbioses in corals’ ability to thrive in multiple dynamic environments and potential role in connectivity, few studies have focused on mesophotic Symbiodinium assemblages. This study examines these assemblages in Montastraea cavernosa found at shallow (20–25 m) and mesophotic (45–50 m) depths at Flower Garden Banks National Marine Sanctuary and McGrail Bank, in the northwest Gulf of Mexico. Mesophotic corals contained significantly more Symbiodinium cells, more chlorophyll a per Symbiodinium cell, and more chlorophyll a and c2 per unit area coral tissue than shallow corals. However, both mesophotic and shallow M. cavernosa contained similar chlorophyll c2 per Symbiodinium cell. Next-generation sequencing of the internal transcribed spacer region (ITS2) of the ribosomal DNA indicated similar Symbiodinium assemblage diversity at all banks and between depths. All assemblages were dominated by sequences most closely related to S. goreaui, type C1, with three additional low-abundance sequences, identified as 2 C types and 1 A type, also consistently observed among colonies. Both the dominant C1 sequence and the background sequences persisted over two sampling years. These results suggest that algal symbiont assemblages will not limit connectivity potential in M. cavernosa in the northwest Gulf of Mexico. Furthermore, we hypothesize that increased Symbiodinium abundance may represent an effective light-harvesting strategy on light-limited mesophotic coral reefs.

Circadian and irradiance effects on expression of antenna protein genes and pigment contents in dinoflagellate Prorocentrum donghaiense (Dinophycae)

PCP and acpPC are the two major antennae proteins that bind pigments in peridinin-containing dinoflagellates. The relationship between antennae proteins and cellular pigments at molecular level is still poorly understood. Here we identified and characterized the two antennae protein genes in dinoflagellate Prorocentrum donghaiense under different light conditions. The mature PCP protein was 32 kDa, while acpPC was a polyprotein each of 19 kDa. Both genes showed higher expression under low light than under high light, suggesting their possible role in a low light adaptation mechanism. The two genes showed differential diel expression rhythm, with PCP being more highly expressed in the dark than in the light period and acpPC the other way around. HPLC analysis of cellular pigments indicated a diel change of chlorophyll c2, but invariability of other pigments. A stable peridinin: chlorophyll a pigment ratio was detected under different light intensities and over the diel cycle, although the diadinoxanthin:chlorophyll a ratio increased significantly with light intensity. The results suggest that 1) PCP and acpPC genes are functionally distinct, 2) PCP and acpPC can function under low light as an adaptive mechanism in P. donghaiense, 3). the ratios of diadinoxanthin:chlorophyll a and peridinin: chlorophyll a can potentially be used as an indicator of algal photophysiological status and a pigment signature respectively under different light conditions in P. donghaiense.

Antimelanoma activity of Heterocapsa triquetra pigments

Heterocapsa triquetra (Ht), a non-toxic dinophyte, was selected to investigate the presence of pigments inhibiting melanoma cells growth. We developed an innovative microwave-assisted extraction process (MAE), determined the Ht pigment composition using Reverse Phase-High Performance Liquid Chromatography (HPLC) and high-resolution Ultra Performance Liquid Chromatography-Mass Spectrometry E (UPLC-MSE) analysis and assessed their antiproliferative activity in human invasive melanoma cells (A2058) grown in vitro. MAE allowed a fast and efficient extraction of Ht pigments as compared with conventional processes (soaking, sonication). Seventeen pigments and derivatives were unequivocally identified by UPLC-MSᴱ in the Ht extract, among which two were supposed to be produced during the extraction (hydroxylated chlorophyll a and pheophytin a). Pigment composition was fully coherent with that usually described for the DINO-1 peridiniales order. It included the uncommon carotenoids P457, peridininol, peridinin, pyrrhoxanthin, diadinochrome and diadinoxanthin. Ht pigment ethanol extract induced 18% growth inhibition of A2058 malignant melanoma cell line after a 72h treatment at 100μg·mL−1 and pigment purification confirmed the highly significant antiproliferative activity of chlorophyll c2, peridinin, dinoxanthin and diatoxanthin on this cell line. Our data demonstrate for the first time the effectiveness of microwave irradiation to extract pigments from dinophytes. They also indicate that Heterocapsa triquetra and other dinophytes are interesting sources to purify bioactive pigments with antiproliferative activities in chemoresistant melanoma cells. These results strongly support previous findings about the anticancer activity of phytoplankton pigments and reinforce the possible interest of these molecules as natural cytostatic drugs.

Comprehensive chlorophyll composition in the main edible seaweeds

Natural chlorophylls present in seaweeds have been studied regarding their biological activities and health benefit effects. However, detailed studies regarding characterization of the complete chlorophyll profile either qualitatively and quantitatively are scarce. This work deals with the comprehensive spectrometric study of the chlorophyll derivatives present in the five main coloured edible seaweeds. The novel complete MS2 characterization of five chlorophyll derivatives: chlorophyll c2, chlorophyll c1, purpurin-18 a, pheophytin d and phytyl-purpurin-18 a has allowed to obtain fragmentation patterns associated with their different structural features. New chlorophyll derivatives have been identified and quantified by first time in red, green and brown seaweeds, including some oxidative structures. Quantitative data of the chlorophyll content comes to achieve significant information for food composition databases in bioactive compounds.