Daily changes in the phosphoproteome of the dinoflagellate Lingulodinium

The dinoflagellate Lingulodinium has a large number of daily rhythms, many of which have no biochemical correlates. We examined the possibility that changes in protein phosphorylation may mediate some of the rhythmic changes by comparing proteins prepared from midday (LD6) and midnight (LD18) cultures. We used two different methods, one a 2D gel protocol in which phosphoproteins were identified after staining with ProQ Diamond, and the other an LC-MS/MS identification of tryptic phosphopeptides that had been purified by TiO(2) chromatography. Two differentially phosphorylated proteins, a light harvesting complex protein and Rad24, were identified using the 2D gel protocol. Six differentially phosphorylated proteins, a polyketide synthase, an uncharacterized transporter, a LIM (actin binding) domain and three RNA binding domain proteins, were identified using the phosphopeptide enrichment protocol. We conclude that changes in protein phosphorylation may underlie some of the rhythmic behavior of Lingulodinium.     

Rampant gene loss in the underground orchid Rhizanthella gardneri highlights evolutionary constraints on plastid genomes

Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on photosynthesis for energy and have retained ～110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer.     

5 The origin of the dinoflagellate plastid

The Dasycladales is an ancient order of tropical benthic marine green algae, unique in their radially arranged unicellular thalli and well-preserved fossil record due to extensive calcification of the thallus. The inference of an accurate phylogeny for the Dasycladales is important in order to better understand stratigraphy, character evolution, and classification. Previous analyses ( rbc L and 18S rDNA) suggested that the Family Acetabulariaceae is monophyletic, but that the Family Dasycladaceae is a basal paraphyletic assemblage. However, the two data sets disagreed regarding genus- and species-level relationships within the Dasycladales. For example, the placement of the genera, Halicoryne , Bornetella and Cymopolia were incongruent. Given the conflicting results of these previous analyses, the current project examined a third highly conserved nuclear-encoded gene, 26S rDNA. Aligned 26S rDNA sequences were analyzed with parsimony and model-based methods and compared to previous results based on18S and rbc L sequences. Family-level relationships based on 26S rDNA were congruent with previous studies: the Acetabulariaceae is monophyletic while the Dasycladaceae is paraphyletic. In addition, acetabulariacean genera are not monophyletic, suggesting that the presence of a corona inferior or calcification of gametes may not be appropriate to define genera. Within the Dasycladaceae, the basal position of Cymopolia is supported by 26S rDNA, a result congruent with rbcL and stratigraphy but not with 18S data. These results will be discussed in the context of morphological character evolution, fossil stratigraphy and family, tribal and generic relationships among these living algal fossils. Supported in part by NSF grant DEB-0128977 to FWZ.     

Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum

Plastid DNA was purified from the dinoflagellate Amphidinium operculatum. The genes atpB, petD, psaA, psbA and psbB have been shown to reside on single-gene minicircles of a uniform size of 2.3–2.4 kb. The psaA and psbB genes lack conventional initiation codons in the expected positions, and may use GTA for translation initiation. There are marked biases in codon preference. The predicted PsbA protein lacks the C-terminal extension which is present in all other photosynthetic organisms except Euglena gracilis, and there are other anomalies elsewhere in the predicted amino acid sequences. The non-coding regions of the minicircles contain a “core” region which includes a number of stretches that are highly conserved across all minicircles and modular regions that are conserved within subsets of the minicircles. Received: 8 September 1999 / Accepted: 10 November 1999     

S-phase and M-phase timing are under independent circadian control in the dinoflagellate Lingulodinium

In many phytoplankton species, cell division (mitosis) usually occurs at defined times of day. This timing is also observed under constant conditions, indicating that it is regulated by a circadian clock rather than by a simple response to the light-dark cycle. For those algae with cell cycles longer than a day, the clock opens a window of opportunity for mitosis at a particular time of day through which cells in an appropriate phase of the cell cycle can pass. Although the timing of mitosis is generally studied due to ease of measurement, for some phytoplankton the timing of S-phase is also circadian. This thus raises the possibility that mitosis is not directly gated by the clock but occurs instead at a defined interval (a constant G2 length) following a circadian controlled S-phase. To determine if the clock exercises independent control over the timing of both S- and M-phase, we measured the timing of both S- and M-phase in cultures of the dinoflagellate Lingulodinium grown under a variety of different photoperiods. We interpret the phase angles of both rhythms, in particular those resulting in a change in the length of G2, as an indication that the clock independently regulates the timing of S-phase and mitosis.     

Circadian photosynthetic reductant flow in the dinoflagellate Lingulodinium is limited by carbon availability

Circadian rhythms are the observed outputs of endogenous daily clocks and are thought to provide a selective advantage to cells adapted to daily light/dark cycles. However, the biochemical links between the clock and the overt rhythms in cell physiology are generally not known. Here, we examine the circadian rhythm in O₂ evolution by cultures of the dinoflagellate Lingulodinium, a rhythm previously ascribed to rhythmic electron flow through photosystem II. We find that O₂ evolution rates increase when CO₂ concentrations are increased, either following addition of DIC or a rapid decrease in culture pH. In medium containing only nitrate as an electron acceptor, O₂ evolution rates mirror the circadian rhythm of nitrate reductase activity in the cells. Furthermore, competition between photosynthetic electron flow to carbon and to nitrate varies in its relative efficiency through the day–night cycle. We also find, using simultaneous and continuous monitoring of pH and O₂ evolution rates over several days, that while culture pH is normally rhythmic, circadian changes in rates of O₂ evolution depend not on the external pH but on levels of internal electron acceptors. We propose that the photosynthetic electron transport rhythm in Lingulodinium is driven by the availability of a reductant sink.     

Genome evolution of a tertiary dinoflagellate plastid

The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata.     

Migration of the plastid genome to the nucleus in a peridinin dinoflagellate

Dinoflagellate algae are important primary producers and of significant ecological and economic impact because of their ability to form "red tides". They are also models for evolutionary research because of an unparalleled ability to capture photosynthetic organelles (plastids) through endosymbiosis. The nature and extent of the plastid genome in the dominant perdinin-containing dinoflagellates remain, however, two of the most intriguing issues in plastid evolution. The plastid genome in these taxa is reduced to single-gene minicircles encoding an incomplete (until now 15) set of plastid proteins. The location of the remaining photosynthetic genes is unknown. We generated a data set of 6,480 unique expressed sequence tags (ESTs) from the toxic dinoflagellate Alexandrium tamarense (for details, see the Experimental Procedures in the Supplemental Data) to find the missing plastid genes and to understand the impact of endosymbiosis on genome evolution. Here we identify 48 of the non-minicircle-encoded photosynthetic genes in the nuclear genome of A. tamarense, accounting for the majority of the photosystem. Fifteen genes that are always found on the plastid genome of other algae and plants have been transferred to the nucleus in A. tamarense. The plastid-targeted genes have red and green algal origins. These results highlight the unique position of dinoflagellates as the champions of plastid gene transfer to the nucleus among photosynthetic eukaryotes.     

Hydrogen peroxide and nitric oxide trigger redox-related cyst formation in cultures of the dinoflagellate Lingulodinium polyedrum

The dinoflagellate Lingulodinium polyedrum is a toxin producer that shows the ability of turning to resting cysts as a survival strategy when exposed to environmental unfavorable conditions, such as nitrogen and phosphorus depletion, abrupt changes in temperature or light, and chemical or mechanical stress. Algal adaptation to all these conditions involves hydrogen peroxide (H₂O₂) and nitric oxide (NO) as key redox signals for housekeeping cellular processes. Thus, we aim here to shed light on the role of H₂O₂ and NO (from aqueous decomposition of sodium nitroprusside, SNP) as prooxidant agents and putative redox signals for encystment of the dinoflagellate L. polyedrum. Harsh oxidative stress imposed by 500 μM H₂O₂ treatment forced L. polyedrum cells to rapidly encyst, in less than 30 min, whereas slower cyst formation was observed upon lower H₂O₂ doses. L. polyedrum encystment was marked by a significant increase in the antioxidant carotenoid peridinin, although other photosynthetic pigments (chlorophyll a and β-carotene) and light-harvesting complexes (peridinin complex protein, PCP) were all diminished in cyst forms. Although SOD activity (a frontline antioxidant enzyme) was severely inhibited by increasing doses of H₂O₂, a theoretical compensatory effect was provided by the dose-dependent increase of ascorbate peroxidase activity (APX), which resulted in significant lower levels of lipid peroxidation during cyst formation. Although SNP data cannot be fully compared to those found with H₂O₂ treatments, changes in APX activity and in biomarkers of lipid and protein oxidation matched the dose–responses found in H₂O₂ experiments, revealing similar biochemical and morphological responses against increasing oxidative conditions during cyst formation. Our data significantly contribute to a better understanding of the relationship between encystment, photosynthesis, and antioxidant responses triggered by H₂O₂ and NO in L. polyedrum, a harmful diarrhetic shellfish poisoning toxin (DSPs) producer.     

Selective feeding and foreign plastid retention in an Antarctic dinoflagellate

The peridinin‐containing plastid found in most photosynthetic dinoflagellates is thought to have been replaced in a few lineages by plastids of chlorophyte, diatom, or haptophyte origin. Other distinct lineages of phagotrophic dinoflagellates retain functional plastids obtained from algal prey for different durations and with varying source species specificity. 18S rRNA gene sequence analyses have placed a novel gymnodinoid dinoflagellate isolated from the Ross Sea (RSD) in the Kareniaceae, a family of dinoflagellates with permanent plastids of haptophyte origin. In contrast to other species in this family, the RSD contains kleptoplastids sequestered from its prey, Phaeocystis antarctica. Culture experiments were employed to determine whether the RSD fed selectively on P. antarctica when offered in combination with another polar haptophyte or cryptophyte species, and whether the RSD, isolated from its prey and starved, would take up plastids from P. antarctica or from other polar haptophyte or cryptophyte species. Evidence was obtained for selective feeding on P. antarctica, plastid uptake from P. antarctica, and increased RSD growth in the presence of P. antarctica. The presence of a peduncle‐like structure in the RSD suggests that kleptoplasts are obtained by myzocytosis. RSD cells incubated without P. antarctica were capable of survival for at least 29.5 months. This remarkable longevity of the RSD's kleptoplasts and its species specificity for prey and plastid source is consistent with its prolonged co‐evolution with P. antarctica. It may also reflect the presence of a plastid protein import mechanism and genes transferred to the dinokaryon from a lost permanent haptophyte plastid.     

Circadian rhythm of a TCA cycle enzyme is apparently regulated at the translational level in the dinoflagellate Lingulodinium polyedrum

Previously, the authors have reported that intracellular amounts of several metabolic-related enzymes from the photosynthetic dinoflagellate Lingulodinium polyedrum(formerly Gonyaulax polyedra) showed a daily rhythm under a 12:12 h LD cycle. This led the authors to hypothesize that a circadian clock controls metabolism, including the tricarboxylic acid (TCA) cycle. In this study, the authors investigated daily changes in the levels of mRNA, protein, and enzyme activity of several metabolic enzymes during 12:12 h LD, 8:16 h LD, and constant light conditions. The NADP-dependent isocitrate dehydrogenase (NADPICDH) in the TCA cycle exhibited circadian changes of protein abundance and enzyme activity under all conditions, whereas its mRNA level remained constant throughout the cycle. These results indicate that the rhythm of NADPICDH is regulated by a circadian control of protein synthesis or modification rather than by message levels and suggest that the TCA cycle may be controlled by the circadian clock system.     

Identification and transcription of transfer RNA genes in dinoflagellate plastid minicircles

The dinoflagellate chloroplast genome is unique in that the genes are found on small circular DNA molecules carrying from one to three genes. In addition, only 14 of the typical chloroplast-located genes have so far been discovered on minicircles, while a number have been transferred to the nucleus. We have sequenced four new minicircles from the dinoflagellate Heterocapsa triquetra, three of which carry a single protein-coding gene (psbD, psbE, petD) and one that appears to be an "empty" circle. Using the tRNA prediction programs ARAGORN and tRNAscan-SE, tRNA-Met was found in the petD circle immediately downstream of the end of petD, while tRNA-Trp and tRNA-Pro were detected in the psbE and petD circles as well as in several chimeric circles of H. triquetra and the psbA minicircles of Heterocapsa pygmaea. RT-PCR showed that the tRNAs were co-transcribed with the protein-coding genes that preceded them, and cleaved from the precursor before a poly(U) tail was added to the mRNA.     

A deviant genetic code in the green alga-derived plastid in the dinoflagellate Lepidodinium chlorophorum

We here report a deviant genetic code, in which AUA is read as methionine (Met) instead of isoleucine (Ile), in the green alga-derived plastid in the dinoflagellate Lepidodinium chlorophorum. Although L. chlorophorum cDNA sequences of 11 plastid-encoded genes were deposited in the GenBank database, the non-canonical usage of AUA in this dinoflagellate plastid has been overlooked prior to this study. We compared 11 plastid-encoded genes of L. chlorophorum with the corresponding genes of 17 green algal plastids. Intriguingly, AUA often occurred in the L. chlorophorum sequences at codon positions that are predominantly occupied by Met amongst the green algal sequences. Coincidentally, the L. chlorophorum sequences utilized few AUA codons at the positions predominantly occupied by Ile amongst the green algal sequences. These observations clearly indicated that both AUA and AUG encode Met, while AUU and AUC encode Ile, in the L. chlorophorum plastid. Despite the rapidly-evolving nature of L. chlorophorum plastid-encoded genes, our statistical tests incorporating the deviant code suggest no significant difference in amino acid composition among the L. chlorophorum plastid and the green algal plastids considered in this study. Finally, the possible evolutionary events required for the reassignment of AUA from Ile to Met in Lepitodinium plastids were discussed.     

Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis

Current understanding of the plastid proteome comes almost exclusively from studies of plants and red algae. The proteome in these taxa has a relatively simple origin via integration of proteins from a single cyanobacterial primary endosymbiont and the host. However, the most successful algae in marine environments are the chlorophyll c-containing chromalveolates such as diatoms and dinoflagellates that contain a plastid of red algal origin derived via secondary or tertiary endosymbiosis. Virtually nothing is known about the plastid proteome in these taxa. We analyzed expressed sequence tag data from the toxic "Florida red tide" dinoflagellate Karenia brevis that has undergone a tertiary plastid endosymbiosis. Comparative analyses identified 30 nuclear-encoded plastid-targeted proteins in this chromalveolate that originated via endosymbiotic or horizontal gene transfer (HGT) from multiple different sources. We identify a fundamental divide between plant/red algal and chromalveolate plastid proteomes that reflects a history of mixotrophy in the latter group resulting in a highly chimeric proteome. Loss of phagocytosis in the "red" and "green" clades effectively froze their proteomes, whereas chromalveolate lineages retain the ability to engulf prey allowing them to continually recruit new, potentially adaptive genes through subsequent endosymbioses and HGT. One of these genes is an electron transfer protein (plastocyanin) of green algal origin in K. brevis that likely allows this species to thrive under conditions of iron depletion.