Chromophyte Algae Research Articles

Pigment configuration in the light-harvesting protein of the xanthophyte alga Xanthonema debile.

The soil chromophyte alga Xanthonema (X.) debile contains only non-carbonyl carotenoids and Chl-a. X. debile has an antenna system denoted Xanthophyte light-harvesting complex (XLH) that contains the carotenoids diadinoxanthin, heteroxanthin, and vaucheriaxanthin. The XLH pigment stoichiometry was calculated by chromatographic techniques and the pigment-binding structure studied by resonance Raman spectroscopy. The pigment ratio obtained by HPLC was found to be close to 8:1:2:1 Chl-a:heteroxanthin:diadinoxanthin:vaucheriaxanthin. The resonance Raman spectra suggest the presence of 8-10 Chl-a, all of which are 5-coordinated to the central Mg, with 1-3 Chl-a possessing a macrocycle distorted from the relaxed conformation. The three populations of carotenoids are in the all-trans configuration. Vaucheriaxanthin absorbs around 500-530nm, diadinoxanthin at 494nm and heteroxanthin at 487nm at 4.5K. The effective conjugation length of heteroxanthin and diadinoxanthin has been determined as 9.4 in both cases; the environment polarizability of the heteroxanthin and diadinoxanthin binding pockets is 0.270 and 0.305, respectively.

Kingdom Chromista and its eight phyla: a new synthesis emphasising periplastid protein targeting, cytoskeletal and periplastid evolution, and ancient divergences

In 1981 I established kingdom Chromista, distinguished from Plantae because of its more complex chloroplast-associated membrane topology and rigid tubular multipartite ciliary hairs. Plantae originated by converting a cyanobacterium to chloroplasts with Toc/Tic translocons; most evolved cell walls early, thereby losing phagotrophy. Chromists originated by enslaving a phagocytosed red alga, surrounding plastids by two extra membranes, placing them within the endomembrane system, necessitating novel protein import machineries. Early chromists retained phagotrophy, remaining naked and repeatedly reverted to heterotrophy by losing chloroplasts. Therefore, Chromista include secondary phagoheterotrophs (notably ciliates, many dinoflagellates, Opalozoa, Rhizaria, heliozoans) or walled osmotrophs (Pseudofungi, Labyrinthulea), formerly considered protozoa or fungi respectively, plus endoparasites (e.g. Sporozoa) and all chromophyte algae (other dinoflagellates, chromeroids, ochrophytes, haptophytes, cryptophytes). I discuss their origin, evolutionary diversification, and reasons for making chromists one kingdom despite highly divergent cytoskeletons and trophic modes, including improved explanations for periplastid/chloroplast protein targeting, derlin evolution, and ciliary/cytoskeletal diversification. I conjecture that transit-peptide-receptor-mediated ‘endocytosis’ from periplastid membranes generates periplastid vesicles that fuse with the arguably derlin-translocon-containing periplastid reticulum (putative red algal trans-Golgi network homologue; present in all chromophytes except dinoflagellates). I explain chromist origin from ancestral corticates and neokaryotes, reappraising tertiary symbiogenesis; a chromist cytoskeletal synapomorphy, a bypassing microtubule band dextral to both centrioles, favoured multiple axopodial origins. I revise chromist higher classification by transferring rhizarian subphylum Endomyxa from Cercozoa to Retaria; establishing retarian subphylum Ectoreta for Foraminifera plus Radiozoa, apicomonad subclasses, new dinozoan classes Myzodinea (grouping Colpovora gen. n., Psammosa), Endodinea, Sulcodinea, and subclass Karlodinia; and ranking heterokont Gyrista as phylum not superphylum.

Electrostatic interaction of positive charges on the surface of Psb31 with photosystem II in the diatom Chaetoceros gracilis

Psb31, a novel extrinsic protein found in diatom photosystem II (PSII), directly binds to PSII core subunits, independent of the other extrinsic proteins, and functions to maintain optimum oxygen evolution. However, how Psb31 electrostatically interacts with PSII intrinsic proteins remains to be clarified. In this study, we examined electrostatic interaction of Psb31 with PSII complexes isolated from the diatom Chaetoceros gracilis. Positive or negative charges of isolated Psb31 proteins were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME), respectively, resulting in formation of uncharged groups. NSP-modified Psb31 did not bind to PSII with a concomitant increase in NSP concentration, whereas GME-modified Psb31 clearly bound to PSII with retention of oxygen-evolving activity, indicating that positive charges of Lys residues and the N-terminus on the surface of Psb31 are involved in electrostatic interactions with PSII intrinsic proteins. Mass spectrometry analysis of NSP-modified Psb31 and sequence comparisons of Psb31 from C. gracilis with other chromophyte algae led to identification of three Lys residues as possible binding sites to PSII. Based on these findings, together with our previous cross-linking study in diatom PSII and a red algal PSII structure, we discuss binding properties of Psb31 with PSII core proteins.

Variability of chromophytic phytoplankton in the North Pacific Subtropical Gyre

Eukaryotic phytoplankton play important roles in regulating productivity and material export in oligotrophic ocean ecosystems. In this study, we examined the vertical and temporal variability in planktonic Chromalveolate (hereafter chromophyte) assemblages over a 2-year period (2007–2009) at Station ALOHA (22°45′N, 158°W) in the North Pacific Subtropical Gyre (NPSG). Polymerase chain reaction (PCR) amplification, cloning, and sequencing of form ID rbcL genes from samples collected at nearly monthly intervals provided information on the diversity, abundances, and variability associated with chromophytic phytoplankton. Despite persistently oligotrophic conditions, the euphotic zone of this habitat supported a phylogenetically diverse assemblage of chromophytic algae, including representatives of various genera of diatoms, pelagophytes, prymnesiophytes, and dinoflagellates. Quantitative PCR (qPCR) amplification of diatom, prymnesiophyte, and pelagophyte rbcL phylotypes revealed that the population structure of these assemblages was highly variable in time, with gene abundances often varying more than an order of magnitude between successive months. Diatom rbcL genes were typically the most abundant in both the upper and lower regions of the euphotic zone, while rbcL gene abundances of the prymnesiophytes and pelagophytes were significantly greater (One-way ANOVA, P<0.05) in the lower regions of the euphotic zone (75–125m) than in the upper euphotic zone (5–45m). Similarly, we observed elevated concentrations of 19-hexanoxyfucoxanthin and 19-butanoxyfucoxanin (diagnostic pigments of prymnesiophytes and pelagophytes, respectively) in the lower euphotic zone, while concentrations of fucoxanthin (a diagnostic diatom pigment) demonstrated less vertical structure. Analyses of samples collected using sediment traps deployed at 150m revealed that members of diatoms, prymnesiophytes, and pelagophytes all contributed to material export out of the upper ocean. None of the phytoplankton groups displayed significant seasonality in gene abundances or fluxes over the period of observations. Our study confirms that diatoms are ubiquitous and diverse members of the euphotic zone phytoplankton assemblage in the NPSG, while prymnesiophytes and pelagophytes appear to capitalize on the relatively nutrient-enriched but low light conditions characteristic of the deeper euphotic zone waters. The combined use of molecular- and pigment-based tools demonstrated that despite persistently oligotrophic conditions chromophytic plankton are perennial contributors to upper ocean biomass and particle export in the NPSG.

Significant Role for Microbial Autotrophy in the Sequestration of Soil Carbon

Soils were incubated for 80 days in a continuously labeled (14)CO(2) atmosphere to measure the amount of labeled C incorporated into the microbial biomass. Microbial assimilation of (14)C differed between soils and accounted for 0.12% to 0.59% of soil organic carbon (SOC). Assuming a terrestrial area of 1.4 × 10(8) km(2), this represents a potential global sequestration of 0.6 to 4.9 Pg C year(-1). Estimated global C sequestration rates suggest a "missing sink" for carbon of between 2 and 3 Pg C year(-1). To determine whether (14)CO(2) incorporation was mediated by autotrophic microorganisms, the diversity and abundance of CO(2)-fixing bacteria and algae were investigated using clone library sequencing, terminal restriction fragment length polymorphism (T-RFLP), and quantitative PCR (qPCR) of the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbL). Phylogenetic analysis showed that the dominant cbbL-containing bacteria were Azospirillum lipoferum, Rhodopseudomonas palustris, Bradyrhizobium japonicum, Ralstonia eutropha, and cbbL-containing chromophytic algae of the genera Xanthophyta and Bacillariophyta. Multivariate analyses of T-RFLP profiles revealed significant differences in cbbL-containing microbial communities between soils. Differences in cbbL gene diversity were shown to be correlated with differences in SOC content. Bacterial and algal cbbL gene abundances were between 10(6) and 10(8) and 10(3) to 10(5) copies g(-1) soil, respectively. Bacterial cbbL abundance was shown to be positively correlated with RubisCO activity (r = 0.853; P < 0.05), and both cbbL abundance and RubisCO activity were significantly related to the synthesis rates of [(14)C]SOC (r = 0.967 and 0.946, respectively; P < 0.01). These data offer new insights into the importance of microbial autotrophy in terrestrial C cycling.

The stereochemistry of chlorophyll- c3 from the haptophyte Emiliania huxleyi: The (13 2R)-enantiomers of chlorophylls- c are exclusively selected as the photosynthetically active pigments in chromophyte algae

Chlorophyll(Chl)- c pigments in algae, diatoms and some prokaryotes are characterized by the fully conjugated porphyrin π-system as well as the acrylate residue at the 17-position. The precise structural characterization of Chl- c 3 from the haptophyte Emiliania huxleyi was performed. The conformations of the π-conjugated peripheral substituents, the 3-/8-vinyl, 7-methoxycarbonyl and 17-acrylate moieties were evaluated, in a solution, using nuclear Overhauser enhancement correlations and molecular modeling calculations. The rotation of the 17-acrylate residue was considerably restricted, whereas the other three substituents readily rotated at ambient temperature. Moreover, the stereochemistry at the 13 2-position was determined by combination of chiral high-performance liquid chromatography (HPLC) with circular dichroism (CD) spectroscopy. Compared with the CD spectra of the structurally related, synthetic (13 2 R)- and (13 2 S)-protochlorophyllide(PChlide)- a, naturally occurring Chl- c 3 had exclusively the (13 2 R)-configuration. To elucidate this natural selection of a single enantiomer, we analyzed the three major Chl- c pigments (Chl- c 1, c 2 and c 3) in four phylogenetically distinct classes of Chl- c containing algae, i.e., heterokontophyta, dinophyta, cryptophyta and haptophyta using chiral HPLC. All the photosynthetic organisms contained only the (13 2 R)-enantiomerically pure Chls- c, and lacked the corresponding enantiomeric (13 2 S)-forms. Additionally, Chl- c 2 was found in all the organisms as the common Chl- c. These results throw a light on the biosynthesis as well as photosynthetic function of Chl- c pigments: Chl- c 2 is derived from 8-vinyl-PChlide- a by dehydrogenation of the 17-propionate to acrylate residues as generally proposed, and the (13 2 R)-enantiomers of Chls- c function as photosynthetically active, light-harvesting pigments together with the principal Chl- a and carotenoids.

Structure of PSI, PSII and antennae complexes from yellow-green alga Xanthonema debile

Photosynthetic carbon fixation by Chromophytes is one of the significant components of a carbon cycle on the Earth. Their photosynthetic apparatus is different in pigment composition from that of green plants and algae. In this work we report structural maps of photosystem I, photosystem II and light harvesting antenna complexes isolated from a soil chromophytic alga Xanthonema debile (class Xanthophyceae). Electron microscopy of negatively stained preparations followed by single particle analysis revealed that the overall structure of Xanthophytes' PSI and PSII complexes is similar to that known from higher plants or algae. Averaged top-view projections of Xanthophytes' light harvesting antenna complexes (XLH) showed two groups of particles. Smaller ones that correspond to a trimeric form of XLH, bigger particles resemble higher oligomeric form of XLH.

NOVEL PROTEINS COMPRISING THE STRAMENOPILE TRIPARTITE MASTIGONEME IN OCHROMONAS DANICA (CHRYSOPHYCEAE)(1).

Two-dimensional (2-D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine-rich, epithelial growth factor (EGF)-like motifs, potentially involved in protein-protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up-regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.

A novel protein in Photosystem II of a diatom Chaetoceros gracilis is one of the extrinsic proteins located on lumenal side and directly associates with PSII core components

The gene encoding a novel extrinsic protein (Psb31) found in Photosystem II (PSII) of a diatom, Chaetoceros gracilis, was cloned and sequenced. The deduced protein contained three characteristic leader sequences targeted for chloroplast endoplasmic reticulum membrane, chloroplast envelope membrane and thylakoid membrane, indicating that Psb31 is encoded in the nuclear genome and constitutes one of the extrinsic proteins located on the lumenal side. Homologous genes were found in a red alga and chromophytic algae but not in other organisms. Genes encoding the other four extrinsic proteins in C. gracilis PSII were also cloned and sequenced, and their leader sequences were characterized and compared. To search for the nearest neighbor relationship between Psb31 and the other PSII components, we crosslinked the PSII particles with the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and found that Psb31 directly associates with PSII core components through electrostatic interaction, suggesting that the novel Psb31 protein is one of the extrinsic proteins constituting the functional oxygen-evolving complex of C. gracilis PSII.

Root rot disease of legumes caused by Aphanomyces euteiches

The Oomycete genus Aphanomyces houses plant and animal pathogens found in both terrestrial and aquatic habitats. Aphanomyces euteiches Drechs. causes seedling damping off and root rot diseases on many legumes. It is the most devastating pea (Pisum sativum) disease in several countries, causing up to 80% losses each year. This strictly soil-borne pathogen may survive many years in soil and no efficient chemical control is currently available. The only way to control the disease is to avoid cultivating legumes in infested fields for up to 10 years. Although huge research effort has been devoted to the Oomycete genus Phytophthora during the last decade, A. euteiches has received little attention and mechanisms by which it infects its hosts are still unclear. A. euteiches is nevertheless an interesting parasite to study plant-oomycete interactions as it is pathogenic on the model legume Medicago truncatula. This review summarizes knowledge about the main characteristics of A. euteiches and presents research currently developed to find new strategies to control this pathogen and to gain insight into its pathogenicity. Aphanomyces euteiches Drechs belongs to a kingdom of diverse eukaryotic protists named Chromista or Straminipila. It is a member of the class Oomycetes (syn. Peronosporomycetes), which gathers organisms resembling fungi through morphological and physiological traits, but are phylogenically related to diatoms, chromophyte algae and other heterokont protists. The genus Aphanomyces is classified within the order Saprolegniales, family Saprolegniaceae s.l. or Leptolegniaceae. Several legumes were found to be hosts for A. euteiches and this pathogen was isolated from field-grown pea, alfalfa, snap bean, vetch, clover, sweet clover and several weed species. The disease begins with the yellowing of root tissue. At a later stage, infected roots become brown and the hypocotyl darkens at the soil line. The pathogen infects the cortex of primary and lateral roots and oospores are formed within the root tissues. http://www.indexfungorum.org/Names/Names.asp (links to taxonomy data), http://www.eugrainlegumes.org/; http://www.medicago.org/ (links to the European Union 'Grain Legume' Integrated Project).

Variability of phycobiliproteins in cyanobacteria detected by delayed fluorescence excitation spectroscopy and its relevance for determination of phytoplankton composition of natural water samples

Delayed fluorescence (DF) excitation spectroscopy is a fast in vivo method to determine phytoplankton composition in freshwaters using the signature of photosynthetically active pigments which is characteristic in each algal class. Four classes could be distinguished: green algae (including Chlorophyceae, Euglenophyceae and Conjugatophyceae), chromophyte algae (including Bacillariophyceae, Chrysophyceae and Dinophyta), Cryptophyta and cyanobacteria. The DF excitation spectra of various cyanobacteria species were investigated with the aim of improving the reliability of the results obtained by DF. Exposing cyanobacteria to different ambient light conditions did not significantly influence the shape of DF excitation spectra. However, the DF excitation spectra of different cyanobacteria species showed different signatures: a high species-specific variability of the ratio phycocyanin (PC):phycoerithrin (PE):allophycocyanin (APC) derived from DF excitation spectra was observed. This variability has to be taken into account when using DF to determine phytoplankton composition in freshwaters dominated by cyanobacteria. The quality of the fits could be significantly improved by using calibration spectra of cyanobacteria species which are, according to their spectral features, at least similar to those species present in the investigated water body.

Cellulose-synthesizing complexes in some chromophyte algae

Freeze fracture investigations have been carried out to investigate cellulose-synthesizing terminal complexes (TCs) in some chromophyte algae. The xanthophycean alga Botrydiopsis intercedens has TCs that consist of 3–8 diagonal rows of particles in the PF of the plasma membrane. A new type of TC was found in the phaeothamniophycean algae Phaeoschizochlamys mucosa, Phaeothamnion confervicola and Stichogloea doederleinii, where TC particles formed 2–3 linear rows. The TC of the eustigmatophycean alga Pseudocharaciopsis minuta was composed of a single linear row of particles and similar to that of phaeophycean algae so far examined. Each of the TCs found in the present study synthesized thin, ribbon-like cellulose microfibrils. A possible pathway of TC development in Botrydiopsis intercedens and TC evolution in the chromophytes are discussed.

Geochemical rate-RNA integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs.

A pilot field experiment to assess the relationship between traditional biogeochemical rate measurements and transcriptional activity of microbial populations was carried out at the LEO 15 site off Tuckerton, N.J. Here, we report the relationship between photosynthetic capacity of autotrophic plankton and transcriptional activity of the large subunit gene (rbcL) for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for primary carbon fixation during photosynthesis. Similar diel patterns of carbon fixation and rbcL gene expression were observed in three of four time series, with maxima for photosynthetic capacity (P(max)) and rbcL mRNA occurring between 10 a.m. and 1 p.m. The lowest P(max) and rbcL levels were detected between 6 p.m. and 10:30 p.m. A significant correlation was found between P(max) and form ID rbcL mRNA (R(2) = 0.56) and forms IA and IB (R(2) = 0.41 and 0.47, respectively). The correlation between the abundance of "diatom" rbcL and P(max) mRNA was modest (R(2) = 0.49; n = 12) but improved dramatically (R(2) = 0.97; n = 10) upon removal of two outliers which represented afternoon samples with high P(max) but lower mRNA levels. Clone libraries from reverse transcription-PCR-amplified rbcL mRNA indicated the presence of several chromophytic algae (diatoms, prymnesiophytes, and chrysophytes) and some eukaryotic green flagellates. Analogous results were obtained from amplified small rRNA sequences and secondary pigment analysis. These results suggest that diatoms were a major contributor to carbon fixation at LEO 15 at the time of sampling and that photosynthetic carbon fixation was partially controlled by transcriptional regulation of the RubisCO gene.

Diverse Eukaryotes have Retained Mitochondrial Homologues of the Bacterial Division Protein FtsZ

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

Food quality controls reproduction of the zebra mussel (Dreissena polymorpha).

Species such as Dreissena polymorpha sometimes contribute substantially in the transfer of primary to secondary production. During the ontogenetic cycle, the reproductive investment of adult mussels is one of the main parameters that affect recruitment success. We studied how food quality and temperature affect the reproductive investment in term of egg mass of D. polymorpha in a lake by sampling mussels monthly from 4 m and 15 m depths. Temperature affected reproduction directly and also indirectly through the food. To assess whether temperature and/or food conditions led to the differences observed in mussels sampled from the two depths, mussels were reared in the laboratory under two different temperature regimes for 3 months, simulating the temperature of the lake at 4 m and 15 m depth. Possible effects of food quality were tested at each temperature using four diets differing in fatty acid composition. Temperature played an important role as a trigger for spawning, and the type of diet clearly affected the reproductive investment. When the heterokont chromophyte alga Nannochloropsis limnetica, which is rich in polyunsaturated fatty acids (PUFAs) and long-chained PUFAs (>C18), was fed to mussels, an increased egg mass was obtained. This result was in contrast to that found when the green alga Scenedesmus obliquus and the cyanobacterium Aphanothece sp., both of which are deficient in long-chained PUFAs, were offered as food to the mussels. Such a PUFA-dependent food quality may affect reproduction in lakes. Food quality effects vary seasonally in a lake and may be most important in summer, when low-food-quality green algae and cyanobacteria are abundant. The low biochemical quality of these blooms may affect at least the later period of gametogenesis of D. polymorpha, which reproduces from June to August.

In vivo characterization of diatom multipartite plastid targeting signals.

Plastids of diatoms and related algae are delineated by four membranes: the outermost membrane (CER) is continuous with the endoplasmic reticulum while the inner two membranes are homologous to plastid envelope membranes of vascular plants and green algae. Proteins are transported into these plastids by pre-sequences that have two recognizable domains. To characterize targeting of polypeptides within diatom cells, we generated constructs encoding green fluorecent protein (GFP) fused to leader sequences. A fusion of GFP to the pre-sequence of BiP [an endoplasmic reticulum (ER)-localized chaperone] resulted in accumulation of GFP within the ER; a construct encoding the pre-sequence of a plastid protein fused to GFP was directed into the plastids. Additional constructs demonstrated that the N-terminal region of the bipartite plastid targeting pre-sequence was necessary for transport of polypeptides to the lumen of the ER, while the C-terminal region was shown to enable the proteins to traverse the plastid double envelope membrane. Our data strongly support the hypothesis of a multi-step plastid targeting process in chromophytic algae and raises questions about the continuity of the ER and CER and the function of the latter in polypeptide trafficking.

Synthesis of several light-harvesting complex I polypeptides is blocked by cycloheximide in symbiotic chloroplasts in the sea slug, Elysia chlorotica (Gould): a case for horizontal gene transfer between alga and animal?

The chloroplast symbiosis between the ascoglossan (=Sacoglossa) sea slug Elysia chlorotica and plastids from the chromophytic alga Vaucheria litorea is the longest-lived relationship of its kind known, lasting up to 9 months. During this time, the plastids continue to photosynthesize in the absence of the algal nucleus at rates sufficient to meet the nutritional needs of the slugs. We have previously demonstrated that the synthesis of photosynthetic proteins occurs while the plastids reside within the diverticular cells of the slug. Here, we have identified several of these synthesized proteins as belonging to the nuclear-encoded family of polypeptides known as light-harvesting complex I (LHCI). The synthesis of LHCI is blocked by the cytosolic ribosomal inhibitor cycloheximide and proceeds in the presence of chloramphenicol, a plastid ribosome inhibitor, indicating that the gene encoding LHCI resides in the nuclear DNA of the slug. These results suggest that a horizontal transfer of the LHCI gene from the alga to the slug has taken place.

Evidence that UV-B tolerance of the photosynthetic apparatus in microalgae is related to the D1-turnover mediated repair cycle in vivo

The present study was conceived to elucidate the potential importance of the D1 turnover-mediated repair mechanism in UV-B tolerance of the photosynthetic apparatus in microalgae. To this end, the lab-identified UV-B sensitive and tolerant species of Chlorophyte and Chromophyte algae was used to examine photosynthetic response to UV-B exposure in the presence vs. the absence of streptomycin, an inhibitor of chloroplast protein synthesis. Measurements of photosynthetic O2 evolution capacity and chlorophyll fluorescence parameters (Fv/Fm, ΦPSII) illustrated species-specific UV-B sensitivity of the photosynthetic apparatus. Addition of the inhibitor streptomycin caused significant enhancements of UV-B-caused depression of photosynthesis in UV-B tolerant species, while little effect was observed in the sensitive species. In the tolerant species, recovery from UV-B induced 20 percnt; decline in Fv/Fm reached completion within 2 hours, much faster than that in the sensitive species. Immunoblotting revealed that exposure to UV-B radiation caused substantial degradation of the D1 protein in the sensitive Heterococcus brevicellularis, which was little enhanced by addition of the inhibitor. The same UV-B exposure lead to less D1 degradation in the tolerant Scenedesmus sp., which was significantly enhanced by addition of the inhibitor. This study shows that UV-B tolerance of the photosynthetic apparatus in microalgae was associated with a strong capacity for recovery from the UV-B-induced damage and this capacity related to the D1 turnover-mediated repair cycle, and largely determined UV-B tolerance of the photosynthetic apparatus in these organisms.

IN VIVO CHARACTERIZATION OF THE DIATOM MULTIPARTATE PLASTID TARGETING SIGNAL

Diatoms and related algae have plastids that are surrounded by four membranes. The outer two membranes are continuous with the endoplasmic reticulum and the inner two membranes are analogous to the plastid envelope membranes of higher plants and green algae. Thus the plastids are completely compartmentalized within the ER membranes. The targeting presequences for nuclear‐encoded plastid proteins have two recognizable domains. The first domain is a classic signal sequence, which presumably targets the proteins to the endoplasmic reticulum. The second domain has characteristics of a transit peptide, which targets proteins to the plastids of higher plants. To characterize these targeting domains, the presequence from the nuclear‐encoded plastid protein AtpC was utilized. A series of deletions of this presequence were fused to Green Fluorescent Protein (GFP) and transformed into cells of the diatom, Phaeodactylum tricornutum. The intracelluar localization of GFP was visualized by fluorescence microscopy. This work demonstrates that the first domain of the presequence is responsible for targeting proteins to the ER lumen and is the essential first step in the plastid protein import process. The second domain is responsible to directing proteins from the ER and through the plastid envelope and only a short portion of the transit peptide‐like domain is necessary to complete this second processing step. In vivo data generated from this study in a fully homologous transformation system has confirmed Gibbs' hypothesis regarding a multistep import process for plastid proteins in chromophytic algae.

Mollusc-algal chloroplast endosymbiosis. Photosynthesis, thylakoid protein maintenance, and chloroplast gene expression continue for many months in the absence of the algal nucleus.

Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO(2) fixation for at least 9 months if provided with only light and a source of CO(2). Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome of V. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes.