Cloning and sequence analysis of the partial sequence of the rbcL from Bryopsis hypnoides

The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the outgroup, neighbor-joining method and maximum parsimony method were used respectively to build phylogenetic trees according to the rbcL exon sequence among 13 species that were the typical species of six phyla. Two kinds of trees showed clearly that there were two groups among those species, the green lineage and the non-green lineage. And the relationships of algae in the green lineage were similar in the two trees but those in the non-green lineage were not consistent. Analysis of codon preference indicated that the codon preference of the rbcL exon of Bryopsis hypnoides distinctly differed from that of the relevant sequence of photosynthetic bacteria.

Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

A group of coenocytic marine algae differs from higher plants,whose totipotency depends on an intact cell(or protoplast).Instead,this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals.It is thought that lectins play a key role in the aggregation process.We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides.The lectin was ca.27 kDa with a pI between pH 5 and pH 6.The absence of carbohydrate suggested that the lectin was not a glycoprotein.The hemagglutinating activity(HA) of the lectin was not dependent on the presence of divalent cations and was inhibited by N-Acetylgalactosamine,N-Acetylglucosamine,and the glycoprotein bovine submaxillary mucin.The lectin preferentially agglutinated Gram-negative bacterium.The HA of this lectin was stable between pH 4 to pH 10.Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution(0.5 mg ml-1).Our results suggest that the regeneration of B.hypnoides is mediated by this lectin.We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater(pH 8.0).Given that nigericin dissipates proton gradients across the membrane,we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

Photosynthetic responses of thalli and isolated protoplasts of Bryopsis hypnoides(Bryopsidales,Chlorophyta)during dehydration

Bryopsis kypnoides Lamouroux is a unique intertidal siphonous green alga whose extruded protoplasm can aggregate spontaneously in seawater to form numerous new cells that can develop into mature algal thalli. In this study, the photosynthetic responses during dehydration of both the thalli and protoplasts isolated from B. kypnoides were measured using a Dual-PAM （pulse amplitude modulation）-100 fluorometer. The results show that the photosynthetic rates of B. kypnoides thalli were maintained for an initial period, beyond which continued desiccation resulted in reduced rates of PSI and PSII. However, the photosynthetic performances of the isolated protoplasts dehydrated in air （CO2 concentration 600-700 mg/L） showed a slight increase of Y（II） at 20% water loss, but the rates decreased thereafter with declining water content. When protoplasts were dehydrated in CO2 deficient conditions （CO2 concentration 40-80 mg/L）, the values of Y（II） declined steadily with increased dehydration without an initial rise. These results indicated that the thalli and isolated protoplasts of this alga can utilize CO2 in ambient air effectively, and the photosynthetic performances of the isolated protoplasts were significantly different from that of the thalli during dehydration. Thus the protoplasts may be an excellent system for the study of stress tolerance.

Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

Using shotgun sequencing data, the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao, China). There are two group I introns in the 16S rRNA gene, which is structurally similar to that of Caulerpa sertularioides (Bryopsidales, Chlorophyta). The chloroplast-encoded tufA gene sequence is 1 230 bp long, very AT-rich (61.5%), and is similar to previously published 16S rRNA sequences of bryopsidinean algae. Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae, Halimedineae, and Ostreobidineae are three distinct lineages. These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae. Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however, this clade lacked robust nodal support. Moreover, the phylogenetic tree inferred from rbcL GenBank sequences, combined with the geographical distributions of Bryopsis species, identified a strongly supportive clade for three differently distributed Asian Bryopsis species. The preliminary results suggesting that these organisms are of distinct regional endemism.

Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

Abstract: Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6–11), and the protoplasts aggregated best at pH 8–9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to z\pH-based translocation (δpH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.

Culturing the Segments of Bryopsis hypnoides Lamouroux Thalli Regenerated from Protoplast Aggregations

Bryopsis hypnoides Lamouroux was regenerated In vitro from the protoplasm squeezed out from wild algae and the regenerated Individuals had an advantage over Indlvlduala from the wild In terms of growth. Culturing of segments of thalll also showed that segments from the regenerated algae grew better than those from individuals from the wild. The segment that corresponds to a part of a cell, at least a part of the protoplasm, can develop Into a mature Individual, Including rhlzold and thallus, suggesting that a multlnucleate alga, such as B. hypnoides, Is different from higher plants, whose totlpotancy Is based on an Intact cell （or protoplasm）. Further cultivation of mature Individuals from segments Indicated that the organelles In the thallus had two ways In which they could survive when the alga was on the decline： （Ⅰ） the organelles were transferred Into a ball with a gelatinous envelope through a formed pipe and the ball seemed to be capable of being propagated; and （Ⅱ） the organelles were aggregated In the thallus and then moved to the outside. An Interesting result Is that one organelle aggregation located outside the thallus germinated and developed Into a mature alga, although most organelle aggregations gradually lost vitality and died. The results of the present study reveal that the aggregation of organelles can regenerate a stronger organism than Individuals from the wild owing to the complete exchange of genetic material and may possibly enable organelles to survive in unfavorable surroundings.

Phototropism of Thalli and Rhizoids Developed from the Thallus Segments of Bryopsis hypnoides Lamouroux

Newly regenerated thalli were used to study the phototropism of Bryopsis hypnoides Lamouroux under different qualities of light. Positive phototropism in the thalli and negative phototropiam In the rhizoida of B. hypnoides were investigated and analyzed in terms of bending. Both thaiii and rhlzoids developed from thallus segments exhibited typical tip growth, and their photoreceptive sites for phototroplam were also restricted to the apical hemisphere. The bending curvature of rhizoids and thalli were determined with unilateral lights at various wavelengths and different fluence rates after a fixed duration of Illumination. The trends of bending from the rhizoid and thallus were coincident, which showed that the action spectrum had a large range, from ultraviolet radiation （366.5 nm） to green light （524 nm）. Based on the bending curvatures, blue light had the highest efficiency, while the efficiency of longer wavelengths （〉500 nm） was significantly lower. External Ca^2＋ had no effect on the bending curvature of thalli and rhlzolda. Blue light （440 nm） induced thallus branching from rhizoids, while red light （650 nm） had no such effect. Fast-occurring chloroplast accumulation In the outermost cytoplasmic layer of the blue light （440 nm）-Irradiated region In the rhizoid was observed, from which protrusions （new thalli） arose after 4 h of the onset of illumination, and this action was thought to be driven by the dynamics of actin microfilamenta.