Phototropism in a red alga, Griffithsia pacifica

In the red alga, Griffithsia pacifica, shoot portions of a plantare positively phototropic and rhizoids are negatively phototropic.We have studied the phototropic response of rhizoids which elongateby tip growth. For 45 min after the beginning of unilateralillumination a rhizoid grows straight, then phototropic curvaturebegins and continues rapidly until the rhizoid is growing awayfrom the light. Curvature is 70–80% complete after 3 hr.If the unilateral stimulus is given for a short time (15 min),curvature again begins at 45 min. However, within an additional30–45 min the rhizoid stops growing away from the lightand wanders back towards its original direction of growth. Phototropismis elicited by light of wavelengths from 350 nm to 500 nm; inlight of wavelengths above 550 nm, little, if any, responseoccurs. ¹Present address: Division of Natural Sciences, University ofCalifornia, Santa Cruz, California 95064, U.S.A. (Received December 10, 1976; )     

Chloroplast genome characterization in the red alga Griffithsia pacifica

Summary It has been suggested that cyanobacteria served as the ancestors for rhodophytic algae whose chloroplasts contain chlorophyll a and phycobilins, and that a rhodophyte served as the plastid source for chromophytic plants that contain chlorophylls a and c. Although organellar DNA has been used to assess phylogenetic relatedness among terrestrial plants and green algae whose chloroplasts contain chlorophylls a and b, few data are presently available on the molecular profile of plastid DNA in chromophytes or rhodophytes.In this study the chloroplast genome of the rhodophytic, filamentous alga Griffithsia pacifica has been characterized. DNA was purified from isolated chloroplasts using protease k treatment and sodium dodecyl sulfate lysis followed by density centrifugation in Hoescht-33258 dye-CsCl gradients. Single and double restriction enzyme digests demonstrate that the DNA prepared from purified chloroplasts has a genome size of about 178 kilobase pairs (kb). A restriction map of this chloroplast genome demonstrates that it is circular and, unlike the chloroplast DNA (cpDNA) in most other plants, contains only a single ribosomal DNA operon. DNA was also purified from the mitochondria that co-isolated with chloroplasts. Mitochondrial DNA consists of molecules that range in size from 27 to 350 kb based on restriction endonuclease digestion and electron microscopic analysis.     

Cytological damage to the red alga Griffithsia pacifica from ultraviolet radiation

Continuous exposure for 7–10 days to 60% of ambient levels (sea level at mid-day in December) of UV-A and UV-B radiation caused cytological damage to regenerating fragments of Griffithsia pacifica under laboratory conditions. There was high mortality of individual cells and entire fragments in UV treated filaments. Rhizoid initiation was slower and rhizoids grew more slowly following UV treatment. After 7 days, UV radiated thalli showed chloroplast and nuclear degeneration. In addition, filaments tended to disarticulate so that single or groups of apparently healthy cells were common in the medium. These data suggest that the subtidal habitat of G. pacifica is based in part on lack of tolerance to UV radiation, and that UV protection mechanisms are not inducible or insufficient to prevent the accumulation of damage in this species.     

Isolation of a cell-fusion hormone from Griffithsia pacifica kylin,a red alga

Filaments of Griffithsia pacifica replace dead cells by the process of cell repair. When an intercalary cell is killed, but its cell wall remains intact holding the two halves of the plant together, the cell above it produces a repair rhizoid cell; the cell below it produces a specialized, rhizoid-like repair shoot cell. The repair rhizoid and shoot grow towards each other, meet, and fuse to form a single shoot cell. Evidence from observations of cell repair in vivo has indicated that the repair rhizoid produces a hormone or hormones which induce the production of the repair shoot, maintain the rhizoid-like morphology and growth of the repair shoot, and attract it to the repair rhizoid for fusion. This hormone has been named rhodomorphin. Using an artificial cell-fusion system we show that repair rhizoids and normal rhizoids, but no shoot cell, can induce decapitated filaments to form repair shoot cells. Decapitated filaments form repair shoot cells only when they are exposed to the hormone within 4–6 h after decapitation; after this time they lose their sensitivity to the hormone. A method has been developed for isolating, and assaying for, the cell-fusion hormone. Rhodomorphin retains its activity for several days at room temperature and for at least two years at-16° C.     

Development in the red alga,Griffithsia pacifica: Control by internal and external factors

Summary Development in the red alga Griffithsia pacifica is affected by both external and internal factors. Under 16:8 photoperiods, both cell division and cell elongation show a diurnal rhythm. The rhythm of division persists for at least 7 cycles in continuous light, and can be reset; this indicates that the timing of cell division is controlled by an endogenous rhythm. Both cell division and elongation require light, but the rate of division of apical cells and the rate of cell elongation are both relatively insensitive to either light intensity or photoperiod. In contrast. division in nodal cells, which leads to branch formation, is strongly promoted by high light intensity or long photoperiods. By manipulating the conditions of illumination, one can obtain Griffithsia plants varying from unbranched to highly branched.     

Antibiotics resistance of a red alga,Griffithsia japonica

To identify antibiotics suitable for stable transformation, we tested the resistance of a red alga,Griffithsia japonica Okamura, to four commonly used antibiotics. Very young germlings, with 1;3 cells, that germinated from the tetraspores were cultured for 40 d in a half PES medium containing kanamycin, streptomycin, hygromycin B, or phleomycin.G. japonica was highly sensitive to 1 μg mL^-1of phleomycin and g mL^-1of hygromycin B. However, it was resistant to kanamycin and low levels of streptomycin and hygromycin B. These results suggest that resistance genes for phleomycin or hygromycin can be used as selectable markers for transformation of G.japonica.     

Analysis of expressed sequence tags from the red alga Griffithsia okiensis

Red algae are distributed globally, and the group contains several commercially important species. Griffithsia okiensis is one of the most extensively studied red algal species. In this study, we conducted expressed sequence tag (ESTs) analysis and synonymous codon usage analysis using cultured G. okiensis samples. A total of 1,104 cDNA clones were sequenced using a cDNA library made from samples collected from Dolsan Island, on the southern coast of Korea. The clustering analysis of these sequences allowed for the identification of 1,048 unigene clusters consisting of 36 consensus and 1,012 singleton sequences. BLASTX searches generated 532 significant hits (E-value <10(-4)) and via further Gene Ontology analysis, we constructed a functional classification of 434 unigenes. Our codon usage analysis showed that unigene clusters with more than three ESTs had higher GC contents (76.5%) at the third position of the codons than the singletons. Also, the majority of the optimal codons of G. okiensis and Chondrus crispus belonging to Bangiophycidae were C-ending, whereas those of Porphyra yezoensis belonging to Florideophycidae were G-ending. An orthologous gene search for the P. yezoensis EST database resulted in the identification of 39 unigenes commonly expressed in two rhodophytes, which have putative functions for structural proteins, protein degradation, signal transduction, stress response, and physiological processes. Although experiments have been conducted on a limited scale, this study provides a material basis for the development of microarrays useful for gene expression studies, as well as useful information for the comparative genomic analysis of red algae.     

Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp

Griffithsin (GRFT), a novel anti-HIV protein, was isolated from an aqueous extract of the red alga Griffithsia sp. The 121-amino acid sequence of GRFT has been determined, and biologically active GRFT was subsequently produced by expression of a corresponding DNA sequence in Escherichia coli. Both native and recombinant GRFT displayed potent antiviral activity against laboratory strains and primary isolates of T- and M- tropic HIV-1 with EC50 values ranging from 0.043 to 0.63 nM. GRFT also aborted cell-to-cell fusion and transmission of HIV-1 infection at similar concentrations. High concentrations (e.g. 783 nM) of GRFT were not lethal to any tested host cell types. GRFT blocked CD4-dependent glycoprotein (gp) 120 binding to receptor-expressing cells and bound to viral coat glycoproteins (gp120, gp41, and gp160) in a glycosylation-dependent manner. GRFT preferentially inhibited gp120 binding of the monoclonal antibody (mAb) 2G12, which recognizes a carbohydrate-dependent motif, and the (mAb) 48d, which binds to CD4-induced epitope. In addition, GRFT moderately interfered with the binding of gp120 to sCD4. Further data showed that the binding of GRFT to soluble gp120 was inhibited by the monosaccharides glucose, mannose, and N-acetylglucosamine but not by galactose, xylose, fucose, N-acetylgalactosamine, or sialic acid-containing glycoproteins. Taken together these data suggest that GRFT is a new type of lectin that binds to various viral glycoproteins in a monosaccharide-dependent manner. GRFT could be a potential candidate microbicide to prevent the sexual transmission of HIV and AIDS.     

Actin-mediated chloroplast movement in Griffithsia pacifica (Ceramiales, Rhodophyta)

Chloroplast banding occurs in Griffithsia pacifies Kylin in a daily rhythm. At the start of the light period, chloroplasts have a uniform distribution. During the light period chloroplasts move away from the ends of the cell leaving two chloroplast-free regions by mid-afternoon. Later in the light period these bands disperse as chloroplasts return to the ends of cell where they remain throughout the dark period. After enzyme treatment with β-glucuronidase to permeabilize cell walls, non-banded plants did not form bands after the addition of 5 μg ml^?1 cytochalasin B in dimethyl sulfoxide (DMSO). When enzyme-grown plants with chloroplast bands were treated with cytochalasin B, bands were retained for at least three days. Plants grown in media supplemented with β-glucuronidase or DMSO alone gave banding consistent with untreated controls. Staining of microfilaments with rhodamine-phalloidin before and after treatment with cytochalasin B gave results consistent with chloroplast movement studies. This is the first report of actin-mediated organelle movement in red algae.     

Further Biochemical Characterization of a Cell Fusion Hormone from the Red Alga, Griffithsia pacifica

In the marine red alga, Griffithsia pacifica, the repair processinitiated upon death of an intercalary cell is mediated by aglycoprotein hormone, rhodomorphin. In this paper we show thatthe glycosidic portion of the molecule has terminal -D-mannosylresidues which are at least in part responsible for the bindingof rhodomorphin by concanavalin A. The protein portion of themolecule contains disulfide bridges. These bridges must be intactfor biologically active hormone to be recovered from a denaturedstate. Isolation of active rhodomorphin from SDS-PAGE indicatesa molecular weight of 15,000–17,500. This agrees wellwith our previously published [Watson and Waaland (1983) PlantPhysiol. 71: 327] molecular weight from gel filtration of 14,000. (Received February 26, 1986; Accepted June 2, 1986)     

Cytoplasmic incompatibility following somatic cell fusion inGriffithsia pacifica Kylin,a red alga

Summary Somatic cell fusion between two isolates ofG. pacifica is followed by a cytoplasmic incompatibility reaction (CIR) in the cytoplasm donated by only one of the isolates. This CIR is characterized by the aggregation, fusion and lysis of chloroplasts of the sensitive strain; the chloroplasts of the other strain are unaffected. In addition, the nuclei of both strains retain a normal distribution during the fusion and lysis events. Cell elongation and nuclear division stop in CIR-affected cells. The CIR begins in the hybrid cell and then appears sequentially in adjacent cells of the sensitive strain; this transfer occurs only between living cells which share a crosswall. There is a lag between hybrid cell formation and the initiation of the CIR. This lag is more than 3 times as long at 17 C than at 24 C; over this range, the rate of movement of the CIR along a filament is temperature-insensitive. Thus it appears that a temperature dependent process, perhaps the synthesis of CIR-inducing agents, is required for the initiation of the CIR; subsequent movement of such agents appears to occur by diffusion.Abbreviations CIR Cytoplasmic incompatibility reaction - HC hybrid cell - 1st SC first shoot cell - 2nd SC second shoot cell - 3rd SC third shoot cell - Pac-PP Puerto Penasco isolate - Pac-BC British Columbia isolate This research was supported in part by National Science Foundation Grant PCM 7823240 to SDW; NICHD Developmental Biology Training Grant ST 32 HDO 71835, Traineeship to DJK and a State of Washington Graduate Opportunities for Women and Minorities to DJK.     

Parasexually produced hybrids between female and male plants of Griffithsia tenuis C. Agardh,a red alga

Somatic cell fusion between vegetative cells of a male and a female isolate of Griffithsia tenuis, a marine red alga, has been obtained. Hybrid cells have been isolated and they have regenerated new plants. Almost all these hybrid plants made reproductive structures. In nearly half these cases the first 3–10 cells of the hybrid filament produced reproductive structures chracteristic of the tetrasporic (diploid) phase rather than the sexual (haploid) phase of the life cycle of this alga. However as these filaments continued to grow, cells further along the filament began to produce sexual, either female or male, reproductive structures. The observations suggest that the production of tetrasporangial branches does not require the fusion of male and female nucleic; rather, male and female nucleic remaining separate, act in concert to produce these structures, and in subsequent cell divisions the nuclei of one sex may be left behind allowing the nuclei of the remaining sex to direct the production of sexual branches.     

The wound-healing responses of Antithamnion nipponicum and Griffithsia pacifica (Ceramiales, Rhodophyta) monitored by lectins

The binding of FITC labeled lectins to repair cells of Antithamnion nipponicum Yamada et Inagaki and Griffithsia pacifica Kylin, and their physiological effects on somatic cell fusion have been studied. Results indicate that repair cells strongly bind the lectins ConA and LCA, whereas other lectins did not bind to the cell, The binding of these lectins to the dead cell wall shows ConA and LCA specific substances are secreted from the tip of the repair cells. When fluorescently labeled ConA or LCA was added at various time intervals after wounding, it firstly bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair ceils. Intense labeling at these sites continued throughout the wound-healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion, When added to plants prior to wounding and with continued monitoring, these same lectins were found to act as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. These results suggest that a signal glycoprotein with α-D-mannosyl residues is involved in the wound-healing process of Antithamnion nipponicum. Lectins conjugated with visible tags can be used as a very fast and useful tool to monitor these signal substances.     

Ferredoxin from a red alga, Porphyra umbilicalis.

A plant-algal type ferredoxin was isolated from the red alga, Porphyra umbilicalis. In its oxidised form the ferredoxin had absorption maxima at 277, (281), 323, 420 and 462 nm. Two atoms each of non-haem iron and labile sulphur were present per molecule protein. The midpoint potential of the protein was -400 mV and it effectively mediated electron transport in the NADP-photoreduction system of barley. The amino acid composition of Porphyra umbilicalis ferredoxin was determined as (Lys4, His2, Arg1, Asx10, Thr8, Ser7, Glx16-17, Pro3, Gly7, Ala8, Cys5, Val6, Met1, Ile5, Leu8, Tyr5, Phe2). The minimum molecular weight of approximately 11000 was confirmed by sedimentation-equilibrium studies in the analytical ultracentrifuge. Approaching half of the total amino acid sequence was determined by means of an automatic sequencer.     

Laurequinone,a cyclolaurane sesquiterpene from the red alga Laurencia nidifica

From the red alga Laurencia nidifica a new sesquiterpene of cyclolaurane-type was isolated, and the structure elucidated by spectral analyses and chemical means.     

Aldingenin derivatives from the red alga Laurencia aldingensis

Three brominated bisabolene-type sesquiterpene derivatives, aldingenin B, C and D, together with cholesterol and palmitic acid, have been isolated from the red alga Laurencia aldingensis (Ceramiales, Rodophyta) and their structures elucidated by spectroscopic methods including NMR analysis.