Copper-induced concomitant increases in photosynthesis, respiration, and C, N and S assimilation revealed by transcriptomic analyses in Ulva compressa (Chlorophyta).

BACKGROUND: The marine alga Ulva compressa is the dominant species in copper-polluted coastal areas in northern Chile. It has been shown that the alga tolerates micromolar concentrations of copper and accumulates copper at the intracellular level. Transcriptomic analyses were performed using total RNA of the alga cultivated with 10 µ M copper for 0, 1, 3 and 5 days using RNA-seq in order to identify processes involved in copper tolerance. RESULTS: The levels of transcripts encoding proteins belonging to Light Harvesting Complex II (LHCII), photosystem II (PSII), cytochrome b6f, PSI, LHCI, ATP synthase and proteins involved in repair of PSII and protection of PSI were increased in the alga cultivated with copper. In addition, the level of transcripts encoding proteins of mitochondrial electron transport chain, ATP synthase, and enzymes involved in C, N and S assimilation were also enhanced. The higher percentages of increase in the level of transcripts were mainly observed at days 3 and 5. In contrast, transcripts involved protein synthesis and degradation, signal transduction, and replication and DNA repair, were decreased. In addition, net photosynthesis and respiration increased in the alga cultivated with copper, mainly at days 1 to 3. Furthermore, the activities of enzymes involved in C, N and S assimilation, rubisco, glutamine synthase and cysteine synthase, respectively, were also increased, mainly at days 1 and 3. CONCLUSIONS: The marine alga U. compressa tolerates copper excess through a concomitant increase in expression of proteins involved in photosynthesis, respiration, and C, N and S assimilation, which represents an exceptional mechanism of copper tolerance.

Nuclear genome sequence of the plastid-lacking cryptomonad Goniomonas avonlea provides insights into the evolution of secondary plastids.

BACKGROUND: The evolution of photosynthesis has been a major driver in eukaryotic diversification. Eukaryotes have acquired plastids (chloroplasts) either directly via the engulfment and integration of a photosynthetic cyanobacterium (primary endosymbiosis) or indirectly by engulfing a photosynthetic eukaryote (secondary or tertiary endosymbiosis). The timing and frequency of secondary endosymbiosis during eukaryotic evolution is currently unclear but may be resolved in part by studying cryptomonads, a group of single-celled eukaryotes comprised of both photosynthetic and non-photosynthetic species. While cryptomonads such as Guillardia theta harbor a red algal-derived plastid of secondary endosymbiotic origin, members of the sister group Goniomonadea lack plastids. Here, we present the genome of Goniomonas avonlea-the first for any goniomonad-to address whether Goniomonadea are ancestrally non-photosynthetic or whether they lost a plastid secondarily. RESULTS: We sequenced the nuclear and mitochondrial genomes of Goniomonas avonlea and carried out a comparative analysis of Go. avonlea, Gu. theta, and other cryptomonads. The Go. avonlea genome assembly is ~ 92 Mbp in size, with 33,470 predicted protein-coding genes. Interestingly, some metabolic pathways (e.g., fatty acid biosynthesis) predicted to occur in the plastid and periplastidal compartment of Gu. theta appear to operate in the cytoplasm of Go. avonlea, suggesting that metabolic redundancies were generated during the course of secondary plastid integration. Other cytosolic pathways found in Go. avonlea are not found in Gu. theta, suggesting secondary loss in Gu. theta and other plastid-bearing cryptomonads. Phylogenetic analyses revealed no evidence for algal endosymbiont-derived genes in the Go. avonlea genome. Phylogenomic analyses point to a specific relationship between Cryptista (to which cryptomonads belong) and Archaeplastida. CONCLUSION: We found no convincing genomic or phylogenomic evidence that Go. avonlea evolved from a secondary red algal plastid-bearing ancestor, consistent with goniomonads being ancestrally non-photosynthetic eukaryotes. The Go. avonlea genome sheds light on the physiology of heterotrophic cryptomonads and serves as an important reference point for studying the metabolic "rewiring" that took place during secondary plastid integration in the ancestor of modern-day Cryptophyceae.

Membrane-Based SDS Depletion Ahead of Peptide and Protein Analysis by Mass Spectrometry.

SDS interferes with both bottom-up and top-down MS analysis, requiring removal prior to detection. Filter-aided sample preparation (FASP) is favored for bottom-up proteomics (BUP) while acetone precipitation is popular for top-down proteomics (TDP). We recently demonstrated acetone precipitation in a membrane filter cartridge. Alternatively, our automated electrophoretic device, termed transmembrane electrophoresis (TME), depletes SDS for both TDP and BUP studies. Here TME is compared to these two alternative methods of SDS depletion in both BUP and TDP workflows. To do so, a modified FASP method is described applicable to the SDS purification and recovery of intact proteins, suitable for LC/MS. All three methods reliably deplete >99.8% SDS. TME provide higher sample yields (average 90%) than FASP (55%) or acetone precipitation (57%), translating into higher total protein identifications (973 vs 877 FASP or 890 acetone) and higher spectral matches (2.5 times) per protein. In a top down workflow, each SDS-depletion method yields high-quality MS spectra for intact proteins. These results show each of these membrane-based strategies is capable of depleting SDS with high sample recovery and high spectra quality for both BUP and TDP studies.

Identification of proteins responding to pathogen-infection in the red alga Pyropia yezoensis using iTRAQ quantitative proteomics.

BACKGROUND: Pyropia yezoensis is an important marine crop which, due to its high protein content, is widely used as a seafood in China. Unfortunately, red rot disease, caused by Pythium porphyrae, seriously damages P. yezoensis farms every year in China, Japan, and Korea. Proteomic methods are often used to study the interactions between hosts and pathogens. Therefore, an iTRAQ-based proteomic analysis was used to identify pathogen-responsive proteins following the artificial infection of P. yezoensis with P. porphyrae spores. RESULTS: A total of 762 differentially expressed proteins were identified, of which 378 were up-regulated and 384 were down-regulated following infection. A large amount of these proteins were involved in disease stress, carbohydrate metabolism, cell signaling, chaperone activity, photosynthesis, and energy metabolism, as annotated in the KEGG database. Overall, the data showed that P. yezoensis resists infection by inhibiting photosynthesis, and energy and carbohydrate metabolism pathways, as supported by changes in the expression levels of related proteins. The expression data are available via ProteomeXchange with the identifier PXD009363. CONCLUSIONS: The current data provide an overall summary of the red algae responses to pathogen infection. This study improves our understanding of infection resistance in P. yezoensis, and may help in increasing the breeding of P. porphyrae-infection tolerant macroalgae.

Exploring Cryptic Diversity and Distribution Patterns in the Mallomonas kalinae/rasilis Species Complex with a Description of a New Taxon-Mallomonas furtiva sp. nov.

A complex of closely related Mallomonas taxa belonging to the section Papillosae, M. kalinae Rezácová and M. rasilis Dürrschmidt, has been studied in detail by molecular and morphometric methods. Our investigations uncovered the existence of a new species found in water bodies in Vietnam, which we describe here as Mallomonas furtiva sp. nov. This taxon is morphologically very similar to M. kalinae, from which it differs by minute, but statistically significant morphological differences on the structure of silica scales. Indeed, the principal component analysis of morphological traits measured on silica scales significantly separates all three species in the complex. Mallomonas kalinae and M. furtiva differ by number of papillae on the shield and the dome, as well as by the scale sizes. Likewise, Mallomonas rasilis and M. furtiva are primarily differentiated by the absence of submarginal anterior ribs on silica scales of the former species. Phylogenetic analyses showed that Mallomonas furtiva is closely related to M. kalinae, with which it formed a highly supported lineage. Distribution patterns of all three studied taxa are further discussed.

Colorimetric protein determination in microalgae (Chlorophyta): association of milling and SDS treatment for total protein extraction.

The use of colorimetric methods for protein quantification in microalgae is hindered by their elevated amounts of membrane-embedded intracellular proteins. In this work, the protein content of three species of microalgae was determined by the Lowry method after the cells were dried, ball-milled, and treated with the detergent sodium dodecyl sulfate (SDS). Results demonstrated that the association of milling and SDS treatment resulted in a 3- to 7-fold increase in protein quantification. Milling promoted microalgal disaggregation and cell wall disruption enabling access of the SDS detergent to the microalgal intracellular membrane proteins and their efficient solubilization and quantification.

Three gene phylogeny of the Thoreales (Rhodophyta) reveals high species diversity.

The freshwater red algal order Thoreales has triphasic life history composed of a diminutive diploid "Chantransia" stage, a distinctive macroscopic gametophyte with multi-axial growth and carposporophytes that develop on the gametophyte thallus. This order is comprised of two genera, Thorea and Nemalionopsis. Thorea has been widely reported with numerous species, whereas Nemalionopsis has been more rarely observed with only a few species described. DNA sequences from three loci (rbcL, cox1, and LSU) were used to examine the phylogenetic affinity of specimens collected from geographically distant locations including North America, South America, Europe, Pacific Islands, Southeast Asia, China, and India. Sixteen species of Thorea and two species of Nemalionopsis were recognized. Morphological observations confirmed the distinctness of the two genera and also provided some characters to distinguish species. However, many of the collections were in "Chantransia" stage rather than gametophyte stage, meaning that key diagnostic morphological characters were unavailable. Three new species are proposed primarily based on the DNA sequence data generated in this study, Thorea kokosinga-pueschelii, T. mauitukitukii, and T. quisqueyana. In addition to these newly described species, one DNA sequence from GenBank was not closely associated with other Thorea clades and may represent further diversity in the genus. Two species in Nemalionopsis are recognized, N. shawii and N. parkeri nom. et stat. nov. Thorea harbors more diversity than had been recognized by morphological data alone. Distribution data indicated that Nemalionopsis is common in the Pacific region, whereas Thorea is more globally distributed. Most species of Thorea have a regional distribution, but Thorea hispida appears to be cosmopolitan.

Intraspecific interactions between algae with different nutritional histories.

The diatom Phaeodactylum tricornutum was cultured in five different growth regimes to obtain cells with different composition. Pairs of populations subjected to different treatments were then mixed in a communal culture regime that differed from those of origin. After 6 h, the ratio between the two populations was verified by flow cytometry. Alterations in this ratio were found when cells previously grown at 1 mM NH4+ were mixed with GeO2 - and 0.5 mM NH4+ -grown cells. The nutritional background may thus make cells differently suited to new environmental conditions and afford advantages in terms of reproductive potential. Competitive interactions between populations may result from the differences in the expressed proteome and/or in the availability of tools for regulatory responses. This may have relevance to the persistence of phenotypically neutral variants present in the population best suited to the new condition, after the interaction of the conspecifics with different nutritional histories.

Widely distributed red algae often represent hidden introductions, complexes of cryptic species or species with strong phylogeographic structure.

Despite studies suggesting that most seaweeds are poor dispersers, many red algal species are reported to have circumglobal distributions. Such distributions have mostly been based on morphological identifications, but molecular data have revealed a range of issues with morphologically defined species boundaries. Consequently, the real distribution of such reportedly circumglobal species must be questioned. In this study, we analyzed molecular data sets (rbcL gene) of nine species in the Rhodomelaceae for which samples were available from widely spaced geographical locations. Three overall patterns were identified: (i) species showing strong phylogeographic structure (i.e., phylogenetic similarity correlates with geographical provenance), often to the point that populations from different locations could be considered as different species (Lophosiphonia obscura, Ophidocladus simpliciusculus, Polysiphonia villum, and Xiphosiphonia pinnulata); (ii) species with a broad distribution that is explained, in part, by putative human-mediated transport (Symphyocladia dendroidea and Polysiphonia devoniensis); and (iii) non-monophyletic complexes of cryptic species, most with a more restricted distribution than previously thought (Herposiphonia tenella, Symphyocladia dendroidea, and the Xiphosiphonia pennata complex that includes the species Xiphosiphonia pinnulata and Symphyocladia spinifera). This study shows that widely distributed species are the exception in marine red algae, unless they have been spread by humans.

Molecular phylogeny, systematics, and revision of the type species of Lobomonas, L. francei (Volvocales, Chlorophyta) and closely related taxa.

In the present study, three new strains of the rare volvocalean green alga Lobomonas were isolated from field-collected samples, one from Sardinia (Italy) and two from Argentina, and comparatively studied. The Sardinian and one of the Argentinian strains were identified as Lobomonas francei, the type species of the genus, whereas the second Argentinian strain corresponded to L. panduriformis. Two additional nominal species of Lobomonas from culture collections (L. rostrata and L. sphaerica) were included in the analysis and shown to be morphologically and molecularly identical to the L. francei strains. The presence, number, and shapes of cell wall lobes, the diagnostic criterion of Lobomonas, were shown to be highly variable depending on the chemical composition of the culture medium used. The analyses by SEM gave evidence that the cell wall lobes in Lobomonas originate at the junctions of adjacent cell wall plates by extrusion of gelatinous material. The four L. francei strains had identical nrRNA gene sequences and differed by only one or two substitutions in the ITS1 + ITS2 sequences. In the phylogenetic analyses, L. francei and L. panduriformis were sister taxa; however, another nominal Lobomonas species (L. monstruosa) did not belong to this genus. Lobomonas, together with taxa designated as Vitreochlamys, Tetraspora, and Paulschulzia, formed a monophyletic group that in the combined analyses was sister to the "Chlamydomonas/Volvox-clade." Based on these results, Lobomonas was revised, the diagnosis of the type species emended, a lectotype and an epitype designated, and several taxa synonymized with the type species.

Quantification of Tetradesmus obliquus (Chlorophyceae) cell size and lipid content heterogeneity at single-cell level.

Much of our current knowledge of microbial growth is obtained from studies at a population level. Driven by the realization that processes that operate within a population might influence a population's behavior, we sought to better understand Tetradesmus obliquus (formerly Scenedesmus obliquus) physiology at the cellular level. In this work, an accurate pretreatment method to quantitatively obtain single cells of T. obliquus, a coenobia-forming alga, is described. These single cells were examined by flow cytometry for triacylglycerol (TAG), chlorophyll, and protein content, and their cell sizes were recorded by coulter counter. We quantified heterogeneity of size and TAG content at single-cell level for a population of T. obliquus during a controlled standard batch cultivation. Unexpectedly, variability of TAG content per cell within the population increased throughout the batch run, up to 400 times in the final stage of the batch run, with values ranging from 0.25 to 99 pg · cell-1 . Two subpopulations, classified as having low or high TAG content per cell, were identified. Cell size also increased during batch growth with average values from 36 to 70 µm3  · cell-1 ; yet cell size variability increased only up to 16 times. Cell size and cellular TAG content were not correlated at the single-cell level. Our data show clearly that TAG production is affected by cell-to-cell variation, which suggests that its control and better understanding of the underlying processes may improve the productivity of T. obliquus for industrial processes such as biodiesel production.

Phylogenetic relationships and biogeography of Ptilophora (Gelidiales, Rhodophyta) with descriptions of P. aureolusa, P. malagasya, and P. spongiophila from Madagascar.

The genus Ptilophora currently includes 16 species occurring mostly in subtidal habitats of the Indo-Pacific Ocean, but its global diversity and biogeography are poorly understood. We analyzed mitochondrial cox1, plastid rbcL and plastid psbA sequences from specimens collected in southern Madagascar during the 2010 Atimo Vatae expedition and studied their morphologies. Both morphological and molecular data sets demonstrated the presence of five species in southern Madagascar: Ptilophora hildebrandtii, P. pterocladioides, and three new species described here, P. aureolusa, P. malagasya, and P. spongiophila. Ptilophora aureolusa is distinguished by its compound pinnae with uniformly spaced pinnules. Ptilophora malagasya has an indistinct midrib and irregularly spaced pinnules. Ptilophora spongiophila, heavily coated with sponges, has cylindrical to flattened main axes, lateral and surface proliferations, and spatulate tetrasporangial sori. The species of Ptilophora found in Madagascar are endemic, except P. hildebrandtii, which also occurs in eastern Africa. Ptilophora comprises four phylogenetic groups that map to eastern Australia, Japan, western Australia/Southeast Asia/Madagascar/eastern Africa, and Madagascar/eastern Africa/Aegean Sea. Biogeographical analysis revealed that the ancestor of Ptilophora originated in Australia, but most of the species radiated from Madagascar.

Historical demography and colonization pathways of the widespread intertidal seaweed Hormosira banksii (Phaeophyceae) in southeastern Australia.

The palaeoceanography of southern Australia has been characterized by fluctuating sea levels during glacial periods, changing temperature regimes and modified boundary currents. Previous studies on genetic structuring of species in southeastern Australia have focused mainly on the differentiation of eastern and western populations while the potential role of Bass Strait as a region of overlap for three biogeographic provinces (Peronia, Maugea, and Flindersia) has been largely ignored. This study aimed to explore the likely roles of historic and contemporary factors in determining divergence patterns in the habitat-forming intertidal seaweed Hormosira banksii in southeastern Australia with a special focus on postglacial dispersal into Bass Strait. We examined the genetic diversity of 475 Hormosira specimens collected from 19 sites around southern Australia using DNA sequence analysis of cytochrome oxidase 1. Three major haplotype groups were identified (western, centre and eastern) corresponding with the three existing biogeographical provinces in this region. Historic break points appeared to be retained and reinforced by modern day dispersal barriers. Phylogeographic grouping of Hormosira reflected a combination of historic and contemporary oceanography. As western and eastern group haplotypes were largely absent within Bass Strait, re-colonization after the last glacial maximum appeared to have originated from refuges within or near present day Bass Strait. Patterns of genetic structure for Hormosira are consistent with other marine species in this region and highlight the importance of biogeographical barriers in contributing to modern genetic structure.

Comparative assessment of the Euglena gracilis var. saccharophila variant strain as a producer of the ß-1,3-glucan paramylon under varying light conditions.

Euglena gracilis Z and a "sugar loving" variant strain E. gracilis var. saccharophila were investigated as producers of paramylon, a ß-1,3-glucan polysaccharide with potential medicinal and industrial applications. The strains were grown under diurnal or dark growth conditions on a glucose-yeast extract medium supporting high-level paramylon production. Both strains produced the highest paramylon yields (7.4-8 g · L-1 , respectively) while grown in the dark, but the maximum yield was achieved faster by E. gracilis var. saccharophila (48 h vs. 72 h). The glucose-to-paramylon yield coefficient Ypar/glu  = 0.46 ± 0.03 in the E. gracilis var. saccharophila cultivation, obtained in this study, is the highest reported to date. Proteomic analysis of the metabolic pathways provided molecular clues for the strain behavior observed during cultivation. For example, overexpression of enzymes in the gluconeogenesis/glycolysis pathways including fructokinase-1 and chloroplastic fructose-1,6-bisphosphatase (FBP) may have contributed to the faster rate of paramylon accumulation in E. gracilis var. saccharophila. Differentially expressed proteins in the early steps of chloroplastogenesis pathway including plastid uroporphyrinogen decarboxylases, photoreceptors, and a highly abundant (68-fold increase) plastid transketolase may have provided the E. gracilis var. saccharophila strain an advantage in paramylon production during diurnal cultivations. In conclusion, the variant strain E. gracilis var. saccharophila seems to be well suited for producing large amounts of paramylon. This work has also resulted in the identification of molecular targets for future improvement of paramylon production in E. gracilis, including the FBP and phosophofructokinase 1, the latter being a key regulator of glycolysis.

Phylogeny and species delineation in the marine diatom Pseudo-nitzschia (Bacillariophyta) using cox1, LSU, and ITS2 rRNA genes: A perspective in character evolution.

Analyses of the mitochondrial cox1, the nuclear-encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo-nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I-III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo-nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence-structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.

Morphological diversity and phylogeny of the diatom genus Entomoneis (Bacillariophyta) in marine plankton: six new species from the Adriatic Sea.

The diatom genus Entomoneis is known from the benthos and plankton of marine, brackish, and freshwaters. Entomoneis includes diatoms with a bilobate keel elevated above the valve surface, a sigmoid canal raphe, and numerous girdle bands. Owing mostly to the scarcity of molecular data for a diverse set of species, the phylogeny of Entomoneis has not been investigated in depth. The few previous studies that included Entomoneis were focused on broader questions and the available data were from a small number of either unidentified Entomoneis or well-known species (e.g., E. paludosa). Since the first description of new species combining both molecular and morphological characters (E. tenera), we have continued to cultivate and investigate Entomoneis in the plankton of the Adriatic Sea. Combined multigene phylogeny (SSU rDNA sequences, rbcL, and psbC genes) and morphological observations (LM, SEM and TEM) revealed six new Entomoneis species supported by phylogenetic and morphological data: E. pusilla, E. gracilis, E. vilicicii, E. infula, E. adriatica, and E. umbratica. The most important morphological features for species delineation were cell shape, the degree and mode of torsion, valve apices, the appearance and structure of the transition between keel and valve body, the ultrastructure and the shape of the girdle bands, and the arrangement and density of perforations along the valve and valvocopulae. Our results highlight the underappreciated diversity of Entomoneis and call for a more in-depth morphological and molecular investigation of this genus especially in planktonic habitats.

Two new species of Phyllonema (Rivulariaceae, Cyanobacteria) with an emendation of the genus.

Two untapered, heterocytous species were observed and collected from the intertidal and supratidal zones of the Mexican coastline of the Pacific Ocean near Oaxaca and from the Gulf of Mexico. These populations were highly similar in morphology to the freshwater taxon Petalonema incrustans in the Scytonemataceae. However, 16S rRNA sequence data and phylogenetic analysis indicated that they were sister taxa to the epiphyllic, Brazilian species Phyllonema aveceniicola in the Rivulariaceae, described from culture material. While genetic identity between the two new species was high, they differed significantly in morphology, 16S rRNA gene sequence identity, and sequence and structure of the 16S-23S ITS region. Their morphology differed markedly from the generitype of the previously monotypic Phyllonema, which has tapered, heteropolar, single-false branched trichomes with very thin or absent sheath. The two new species, Phyllonema ansata and Phyllonema tangolundensis, described from both culture and environmental material, have untapered, isopolar, geminately false branched trichomes with thick, lamellated sheaths, differences so significant that the species would not be placed in Phyllonema without molecular corroboration. The morphological differences are so significant that a formal emendation of the genus is required. These taxa provide a challenge to algal taxonomy because the morphological differences are such that one would logically conclude that they represent different genera, but the phylogenetic evidence for including them all in the same genus is conclusive. This conclusion is counter to the current trend in algal taxonomy in which taxa with minor morphological differences have been repeatedly placed in separate genera based primarily upon DNA sequence evidence.

Survey for the presence of a vitronectin-like protein in micro- and macroalgae and cyanobacteria.

Vitronectin (Vn) is a glycoprotein that serves a wide variety of roles in multicellular organisms. It was first identified in multicellular animals but has also been isolated from land plants and some algae, where it appears to serve as an extracellular adhesive molecule. In order to further elucidate presence and localization of a Vn-like protein and its potential role in algae, we surveyed different morphological regions of 24 species of macro- and microalgae and three species of cyanobacteria for the presence of a Vn-like protein. Vn-like proteins were not detected in any of the species of cyanobacteria, microalgae or Rhodophyta investigated. They were detected in several species of the Phaeophyceae and Chlorophyta where their localization was limited to the holdfast and rhizoids of these organisms, respectively. Detection of a Vn-like protein (between 0.0125 and 0.097 µg · µL-1 protein extract) was therefore limited to locations associated with substrate attachment.

Proteomics links the redox state to calcium signaling during bleaching of the scleractinian coral Acropora microphthalma on exposure to high solar irradiance and thermal stress.

Shipboard experiments were each performed over a 2 day period to examine the proteomic response of the symbiotic coral Acropora microphthalma exposed to acute conditions of high temperature/low light or high light/low temperature stress. During these treatments, corals had noticeably bleached. The photosynthetic performance of residual algal endosymbionts was severely impaired but showed signs of recovery in both treatments by the end of the second day. Changes in the coral proteome were determined daily and, using recently available annotated genome sequences, the individual contributions of the coral host and algal endosymbionts could be extracted from these data. Quantitative changes in proteins relevant to redox state and calcium metabolism are presented. Notably, expression of common antioxidant proteins was not detected from the coral host but present in the algal endosymbiont proteome. Possible roles for elevated carbonic anhydrase in the coral host are considered: to restore intracellular pH diminished by loss of photosynthetic activity, to indirectly limit intracellular calcium influx linked with enhanced calmodulin expression to impede late-stage symbiont exocytosis, or to enhance inorganic carbon transport to improve the photosynthetic performance of algal symbionts that remain in hospite. Protein effectors of calcium-dependent exocytosis were present in both symbiotic partners. No caspase-family proteins associated with host cell apoptosis, with exception of the autophagy chaperone HSP70, were detected, suggesting that algal loss and photosynthetic dysfunction under these experimental conditions were not due to host-mediated phytosymbiont destruction. Instead, bleaching occurred by symbiont exocytosis and loss of light-harvesting pigments of algae that remain in hospite. These proteomic data are, therefore, consistent with our premise that coral endosymbionts can mediate their own retention or departure from the coral host, which may manifest as "symbiont shuffling" of Symbiodinium clades in response to environmental stress.

Proteome analysis of cytoplasmatic and plastidic ß-carotene lipid droplets in Dunaliella bardawil.

The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and ß-carotene-rich (ßC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in ßC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The ßC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL heme-binding proteins, phytyl ester synthases, ß-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of ßC-plastoglobuli and the biosynthesis of ß-carotene within ßC-plastoglobuli and hypothesize that ßC-plastoglobuli evolved from eyespot lipid droplets.