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The peculiar distribution of class I and class II aldolases in diatoms and in red algae.

Diatom plastids probably evolved by secondary endocytobiosis from a red alga that was up by a eukaryotic host cell. Apparently, this process increased the complexity of the intracellular distribution of metabolic enzymes. We identified genes encoding fructose-bisphosphate aldolases (FBA) in two centric (Odontella sinensis, Thalassiosira pseudonana) and one pennate (Phaeodactylum tricornutum) diatoms and found that four different aldolases are present in both groups: two plastid targeted class II enzymes (FBAC1 and FBAC2), one cytosolic class II (FBA3) and one cytosolic class I (FBA4) enzyme. The pennate Phaeodactylum possesses an additional plastidic class I enzyme (FBAC5). We verified the classification of the different aldolases in the diatoms by enzymatic characterization of isolated plastids and whole cell extracts. Interestingly, our results imply that in plastids of centric and pennate diatoms mainly either class I or class II aldolases are active. We also identified genes for both class I and class II aldolases in red algal EST databases, thus presenting a fascinating example of the reutilization and recompartmentalization of different aldolase isoenzymes during secondary endocytobiosis but as well demonstrating the limited use of metabolic enzymes as markers for the interpretation of phylogenetic histories in algae.

Aldehyde-Lyases↗

Cloning and characterization of a nuclear gene encoding a starch-branching enzyme from the marine red alga Gracilaria gracilis.

The biosynthesis of starch in red algae occurs in the cytosol, in contrast to green plants where it takes place in the plastid. We have cloned a nuclear gene from the red alga Gracilaria gracilis that encodes a homolog of starch-branching enzymes (SBEs); this gene, which is apparently intron-free, was designated as GgSBE1. A potential TATA box, CAAT boxes, and other potential regulatory elements were observed in its 5' flanking region. The encoded 766-aa peptide shares significant sequence similarity with SBEs from green plants (at least 40%), and with glycogen-branching enzymes (GBEs) from human (46%) and Saccharomyces cerevisiae (45%). Southern-hybridization analysis indicates that the gene is single-copy, although weaker signals suggest that related genes exist in the genome of G. gracilis. Phylogenetic analyses indicate that GgSBE1 groups within the eukaryote branching enzymes (BEs) and not with eubacterial GBEs, suggesting that its gene has not been derived directly from an endosymbiotic cyanobacterium, but instead is ancestrally eukaryotic.

1,4-alpha-Glucan Branching Enzyme↗

Complex group-I introns in nuclear SSU rDNA of red and green algae: evidence of homing-endonuclease pseudogenes in the Bangiophyceae.

The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5' end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors a sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein on the complementary strand. A comparison between related group-I introns in the Bangiophyceae revealed homing-endonuclease-like pseudogenes due to frame-shifts and deletions in Porphyra and Bangia. The Scenedesmus and Porphyra introns provide new insights into the evolution and possible novel functions of nuclear group-I intron proteins.

Amino Acid Motifs↗

Extensibility of isolated cell walls in the giant tip-growing cells of the xanthophycean alga Vaucheria terrestris.

Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.

Algal Proteins↗

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta).

In order to broaden our understanding of the eukaryotic CO2-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO2-concentrating mechanism when grown under limiting CO2 conditions. Using mass-spectrometric measurements of 18O exchange from doubly labelled CO2, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex.

Animals↗

Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga).

Sulfur deprivation in green algae causes reversible inhibition of photosynthetic activity. In the absence of S, rates of photosynthetic O2 evolution drop below those of O2 consumption by respiration. As a consequence, sealed cultures of the green alga Chlamydomonas reinhardtii become anaerobic in the light, induce the "Fe-hydrogenase" pathway of electron transport and photosynthetically produce H2 gas. In the course of such H2-gas production cells consume substantial amounts of internal starch and protein. Such catabolic reactions may sustain, directly or in directly, the H2-production process. Profile analysis of selected photosynthetic proteins showed a precipitous decline in the amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a function of time in S deprivation, a more gradual decline in the level of photosystem (PS) II and PSI proteins, and a change in the composition of the PSII light-harvesting complex (LHC-II). An increase in the level of the enzyme Fe-hydrogenase was noted during the initial stages of S deprivation (0-72 h) followed by a decline in the level of this enzyme during longer (t >72 h) S-deprivation times. Microscopic observations showed distinct morphological changes in C. reinhardtii during S deprivation and H2 production. Ellipsoid-shaped cells (normal photosynthesis) gave way to larger and spherical cell shapes in the initial stages of S deprivation and H2 production, followed by cell mass reductions after longer S-deprivation and H2-production times. It is suggested that, under S-deprivation conditions, electrons derived from a residual PSII H2O-oxidation activity feed into the hydrogenase pathway, thereby contributing to the H2-production process in Chlamydomonas reinhardtii. Interplay between oxygenic photosynthesis, mitochondrial respiration, catabolism of endogenous substrate, and electron transport via the hydrogenase pathway is essential for this light-mediated H2-production process.

Anaerobiosis↗

The role of GlsA in the evolution of asymmetric cell division in the green alga Volvox carteri.

Volvox carteri, a green alga in the order Volvocales, contains two completely differentiated cell types, small motile somatic cells and large reproductive cells called gonidia, that are set apart from each other during embryogenesis by a series of visibly asymmetric cell divisions. Mutational analysis has revealed a class of genes (gonidialess, gls) that are required specifically for asymmetric divisions in V. carteri, but that are dispensable for symmetric divisions. Previously we cloned one of these genes, glsA, and showed that it encodes a chaperone-like protein (GlsA) that has close orthologs in a diverse set of eukaryotes, ranging from fungi to vertebrates and higher plants. In the present study we set out to explore the role of glsA in the evolution of asymmetric division in the volvocine algae by cloning and characterizing a glsA ortholog from one of the simplest members of the group, Chlamydomonas reinhardtii, which does not undergo asymmetric divisions. This ortholog (which we have named gar1, for glsA related) is predicted to encode a protein that is 70% identical to GlsA overall, and that is most closely related to GlsA in the same domains that are most highly conserved between GlsA and its other known orthologs. We report that a gar1 transgene fully complements the glsA mutation in V. carteri, a result that suggests that asymmetric division probably arose through the modification of a gene whose product interacts with GlsA, but not through a modification of glsA itself.

Algal Proteins↗

The antifilarial activity of a marine red alga, Botryocladia leptopoda, against experimental infections with animal and human filariae.

The antifilarial activity of the marine red alga Botryocladia leptopoda against rodent and human lymphatic filarial parasites is described. The animal filarial species included Litomosoides sigmodontis and Acanthocheilonema viteae maintained in cotton rats and Mastomys coucha, respectively, while a subperiodic strain of the human lymphatic filarial parasite Brugia malayi was maintained in M. coucha. The crude extract and its hexane fraction brought about a marked reduction in the peripheral microfilarial level in both of the rodent filarial parasites L. sigmodontis and A. viteae. The microfilaricidal effect began slowly from day 8 or 15 after initiation of treatment and increased with time with a very high efficacy at the end of the observation period against both rodent filariids. The microfilaricidal efficacy was, however, not as prominent in the case of B. malayi. The antifilarial activity, which occurred in the hexane fraction, exerted action at a much lower dose. The product killed a significant proportion of A. viteae and L. sigmodontis adult parasites. In the case of B. malayi, although the macrofilaricidal efficacy was much less than that of the rodent parasites, it (hexane fraction) caused sterilization of a significant proportion of the surviving female parasites. The present findings indicate the possibility of developing an adulticidal and female sterilizing agent against filarial parasites from a marine red alga.

Animals↗

The fucoxanthin-chlorophyll proteins from a chromophyte alga are part of a large multigene family: structural and evolutionary relationships to other light harvesting antennae.

A fucoxanthin-chlorophyll protein (FCP) cDNA from the raphidophyte Heterosigma carterae encodes a 210-amino acid polypeptide that has similarity to other FCPs and to the chlorophyll a/b-binding proteins (CABs) of terrestrial plants and green algae. The putative transit sequence has characteristics that resemble a signal sequence. The Heterosigma fcp genes are part of a large multigene family which includes members encoding at least two significantly different polypeptides (Fcp1, Fcp2). Comparison of the FCP sequences to the recently determined three-dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and the formation of complex-stabilizing ionic interactions are well conserved. Phylogenetic analyses of sequences of light-harvesting proteins shows that the FCPs of several chromophyte phyla form a natural group separate from the intrinisic peridinin-chlorophyll proteins (iPCPs) of the dinoflagellates: Although the FCP and CAB genes shared a common ancestor, these lineages diverged from each other prior to the separation of the CAB LHC I and LHC II sequences in the green algae and terrestrial plants.

Amino Acid Sequence↗

Blue- and green-light signals for gamete release in the brown alga, Silvetia compressa.

The intertidal brown alga Silvetia compressa releases gametes from receptacles (the reproductive tissue) rapidly upon a dark transfer (following a photosynthesis-dependent period in the light, termed potentiation). In this study, the wavelength-dependence of this process was investigated. During the potentiation period in white light (WL), gametes are not released. However, gametes were released during potentiation in blue light (BL), or in low red light/blue light (RL/BL) ratios, but not in RL alone, high RL/BL ratios, or in broadband blue-green light (B-GL) (presence of BL, but absence of RL). RL was as effective as WL for potentiation, i.e., both lead to gamete release following transfer to darkness. Rates of linear photosynthetic electron transport were similar in RL and BL. Gamete release in BL was inhibited by equal amounts of additional narrow-waveband light between the green and red regions of the spectrum, with light-induced gamete release restricted between <491 nm and 509 nm. Very little light-induced gamete release occurred between 530 nm and 650 nm. It is proposed that a BL-responsive photoreceptor is responsible for light-induced gamete release. Transfer of WL-potentiated receptacles to GL near 530 nm resulted in significant de-potentiation and reduced gamete release during a subsequent dark transfer. This effect was not seen at 509 nm or 560 nm and revealed the presence of a second photoreceptor system repressing or counteracting potentiation in the light. We propose that the restriction of gamete release to periods when irradiance is blue-shifted may constitute a depth-sensing mechanism for this intertidal alga, allowing controlled release of gametes at high tide and/or less turbid periods, thus minimizing gamete dilution, and promoting fertilization success.

Fertilization↗

Genotypic variation in tolerance and resistance to fouling in the brown alga Fucus vesiculosus.

In this study, we examined genetic variation in resistance and tolerance to fouling organisms in the brown alga Fucus vesiculosus. We first grew 30 algal genotypes in the field, where we allowed fouling organisms to colonise the genotypes at natural levels. We then conducted a manipulative experiment, where we grew 20 genotypes of algae in aquaria with or without fouling organisms. We measured host resistance as the load of fouling organisms and tolerance as the slope of the regression of algal performance on fouling level. Fouling organisms decreased host growth and contents of phlorotannins and thus have the potential to act as selective agents on algal defenses. We found significant among-genotype variation in both resistance and tolerance to fouling. We did not find a trade-off between resistance and tolerance. We found a marginally significant cost of resistance, but no cost of tolerance. Our results thus indicate that both the tolerance and resistance of F. vesiculosus can evolve as a response to fouling and that the costs of resistance may maintain genetic variation in resistance.

Analysis of Variance↗

Localization of specific monosaccharides in cells of the brown alga Padina gymnospora and the relation to heavy-metal accumulation.

The brown alga Padina gymnospora has been studied due to their ecological significance and biochemical characteristics, including its high capability of heavy-metal accumulation. It has been suggested that the fucans are among the main polysaccharides related to metal binding and precipitation in cell walls. The main purpose of this work was to determine the localization of specific monosaccharides in P. gymnospora cells. In this way, the lectins Ulex europaeus agglutinin and Canavalia ensiformis concanavalin A with specificity to alpha-L-fucose and to terminal residues of alpha-D-glucosyl and alpha-D-mannosyl, respectively, were applied in young individuals. These revealed a preferential distribution of alpha-L-fucose at cell walls near the external surface in cortical cells and near the plasmalemma in cortical and medullar cells. The distribution of alpha-L-fucose in cell walls indicates the distribution of sulfated polysaccharides (sulfated fucans) that colocalize with the heavy-metal granules (Zn and Cd) described in previous works. Therefore, our results suggest that alpha-L-fucose participates in the nucleation and immobilization of heavy metals in P. gymnospora cell walls. An intense labeling of U. europaeus agglutinin and a weak labeling of concanavalin A was also observed in physodes. X-ray microanalysis revealed the presence of zinc, sulfur, and calcium in physodes of algae collected in a heavy-metal-contaminated area. Besides the affinity between polyphenolic compounds and heavy metals, it is suggested that the mechanism of metal binding by physodes could be related to the presence of sulfated fucans.

Cell Wall↗

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: I. The effect of cadmium.

The aim of the study was to elucidate the effect of cadmium ions on the arrangement of the actin and tubulin cytoskeleton, as well as the relationships between cytoskeletal changes and growth processes in the green filamentous alga Spirogyra decimina. Batch cultures of algae were carried out under defined conditions in the presence of various cadmium concentrations. In control cells, the cytoskeleton appeared to be a transversely oriented pattern of both microtubules and actin filaments of various thickness in the cell cortex; colocalization of cortical microtubules and actin filaments was apparent. Microtubules were very sensitive to the presence of cadmium ions. Depending on the cadmium concentration and the time of exposure, microtubules disintegrated into short rod-shaped fragments or they completely disappeared. A steep increase in cell width and a decrease in growth rate accompanied (and probably ensued) a very rapid disintegration of microtubules. Actin filaments were more stable because they were disturbed several hours later than microtubules at any cadmium concentration used. When cadmium ions were washed out, the actin cytoskeleton was rebuilt even in cells in which actin filaments were completely disintegrated at higher cadmium concentrations (40 or 100 microM). The much more sensitive microtubules were regenerated after treatment with lower cadmium concentrations (10 or 15 microM) only.

Actin Cytoskeleton↗

UV-absorbing substance in the red alga Porphyra yezoensis (Bangiales, Rhodophyta) block thymine photodimer production.

The effect of the water-soluble UV-absorbing substance (UVAS) extracted from the marine red alga Porphyra yezoensis Ueda on UV-dependent thymine photodimer production was investigated. The T<>T pyrimidine-pyrimidone 6-4 dimer and the cyclobutane cis-syn T<>T 5-6 dimer produced by UV irradiation with a xenon lamp were analyzed by reverse-phase high-performance liquid chromatography. Although the dimer production was reduced when the irradiation was filtered through a UVAS solution, it decreased more when thymine was mixed with UVAS. Furthermore, UVAS inhibited the degradation of UV-irradiated thymine. The inhibitory effect of UVAS was significantly greater than that of exogenously added adenine or guanine, which forms complementary base pairs with thymine. These data suggest that in addition to its filtering effect against UV radiation, UVAS also protects thymine by a direct molecule-to-molecule energy transfer process. The protective function of UVAS against UV irradiation is advantageous for this alga under strong UV irradiation.

Absorption↗

Salt-regulated mannitol metabolism in algae.

Mannitol, one of the most widely occurring sugar alcohol compounds, is found in bacteria, fungi, algae, and plants. In these organisms the compound acts as a compatible solute and has multiple functions, including osmoregulation, storage, and regeneration of reducing power, and scavenging of active oxygen species. Because of the diverse functions of mannitol, introducing the ability to accumulate it has been a hallmark of attempts to generate highly salt-tolerant transgenic plants. However, transgenic plants have not yet improved significantly in their salt tolerance. Recently, we purified and characterized 2 enzymes that biosynthesize mannitol, mannitol-1-phosphate dehydrogenase (M1PDH) and mannitol-1-phosphate-specific phosphatase, from the marine red alga Caloglossa continua, which grows in estuarine areas where tide levels fluctuate frequently. The activation of Caloglossa M1PDH is unique in that it is regulated by salt concentration at enzyme level. In this review we focus on the metabolism of mannitol, mainly in marine photosynthetic organisms, and suggest how this might be applied to producing salt-tolerant transgenic plants.

Bacteria↗

Variation in storage alpha-polyglucans of red algae: amylose and semi-amylopectin types in Porphyridium and glycogen type in Cyanidium.

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.

Algal Proteins↗

Molecular cloning and antiserum development of cyclin box in the brown tide alga Aureococcus anophagefferens.

Cyclins can be useful cell cycle markers for growth rate studies on harmful algal blooms. In this study, a gene fragment corresponding to cyclin box was cloned for the brown tide alga Aureococcus anophagefferens. This algal gene fragment, designated as Btcycl1, was most similar to cyclin B. Oligopeptides based on the deduced amino acid sequence were synthesized and used to raise an antiserum that reacted on Western blots with a protein of about 63 kDa, the same size as cyclin B in other organisms. The cyclin B-like protein recognized by this antiserum, and the messenger RNA amplified using the primers, were more abundant in exponential cultures and decreased markedly in stationary cultures. This protein also appeared to be cell cycle dependent. Immunofluorescence labeling showed that this antiserum specifically stained a protein in Aureococcus cells and had no cross-reaction with bacteria that were present in the algal culture. The Btcycl1 sequence and the antiserum will provide a useful tool for studies on regulation of in situ growth rate for this brown tide alga.

Journal Article↗

Chemotaxis in the green flagellate alga Chlamydomonas.

Behavior of the green flagellate alga Chlamydomonas changes in response to a number of chemical stimuli. Specific sensitivity of the cells to different substances might appear only at certain stages of the life cycle. The heterogamous species C. allensworthii demonstrates chemotaxis of male gametes towards pheromones excreted by female gametes. In C. reinhardtii chemotaxis towards tryptone occurs only in gametes, whereas chemotaxis towards ammonium, on the contrary, only in vegetative cells. Chemotaxis to different chemical stimuli might involve different mechanisms of reception and signal transduction, elucidation of which has only recently begun. Indirect evidences show that the cells likely respond to tryptone with changes in the membrane electrical conductance. The recently completed project of sequencing the whole nuclear genome of C. reinhardtii provides the basis for future identification of molecular elements of the chemosensory cascade in this alga.

Animals↗