UV-Absorbing Substance in the Red Alga <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (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.     

Comparative study of wild and cultivated <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) based on molecular and morphological data

We compared the wild Porphyra strain OGATSU from northeastern Japan with cultivated Porphyra yezoensis f. narawaensis using the RuBisCO spacer, rbcL, and ITS-1 DNA sequences as well as early gametophyte development. Based on the molecular analyses and detailed morphological observations, OGATSU was identified as P. yezoensis, but also revealed important differences from the cultivated form. Under the same culture conditions, gametophytic blades of OGATSU produced more archeospores than P. yezoensis f. narawaensis strain HG-4. The length of blades and their length-to-width ratios were significantly lower in OGATSU than in HG-4, and the color of OGATSU blades was darker than that of HG-4. The first lateral cell division in conchospore germlings occurred significantly earlier in the OGATSU strain than in the HG-4 strain, resulting in the rounder shape of the OGATSU blade compared to that of P. yezoensis f. narawaensis. These results suggested that wild strains such as OGATSU can provide useful characters that could enhance cultivated varieties in a careful breeding program.     

Identification of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta) meiosis by DNA quantification using confocal laser scanning microscopy

Conchospore germlings of Porphyra yezoensis were stained with a fluorescent dye for DNA and observed with confocal laser scanning microscopy (CLSM). Relative DNA values of the germling nuclei were obtained by measuring fluorescence intensities of nuclear regions of the optically sliced specimens, using the mean value of the smallest blade cells as a reference of the genomic n value. Such quantification revealed that the nuclear DNA amounts of the one-cell, two-cell, and four-cell-stage germlings are approximately 4 × n, 2 × n, and n ∼2 × n values respectively; these values agreed well with the expected ones from the hypothesis that meiosis corresponds to the first successive cell divisions after the conchospore germination. These results are consistent with a previous study on cytogenetic analysis of the chimaera blade formation (Ohme and Miura 1988, Plant Sci 57:135–140) and not consistent with a recent microscopic study (Wang et al. 2006, Phycol Res 54:201–207) which proposed that the first meiotic division occurs at the conchospore formation and the second division at the germination.     

Rapid isolation and sequence analysis of the beta-tubulin gene from <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta)

Beta-tubulin, one of the cytoskeletal proteins, has been highly conserved throughout the evolution of eukaryotes. Degenerate PCR and inverse PCR (iPCR) were used to isolate the full-length beta-tubulin gene and its 5′ and 3′-flanking regions (2799bp) from the marine red alga Porphyra yezoensis. This gene, designated as TubB1, is devoid of introns. The canonical cis-acting elements such as TATA box, CAAT box and polyA signal AAUAAA are not found in flanking sequences, but another putative polyA signal CAYTG is found downstream of the stop codon. Comparison of the deduced 458 amino acid sequences shows higher similarity to the Protoflorideophycidae Cyanidioschyzon merolae (82%) than to the red alga Chondrus crispus (79%). Codon bias indicates strong expression of TubB1. Phylogenetic analysis suggests that the beta-tubulin of P. yezoensis and C. merola go together with fungi and not with green plants. These nucleotide sequence data have been deposited in the DDB/EMBL/Genbank databases under the accession number AY221630.     

Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (Bangiales,Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 ^°C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 ^°C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 ^°C, 23.2± 0.24× 10⁶ protoplasts g⁻¹ fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.     

Genetic Analysis of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Using Microsatellite Markers

Microsatellites are repetitive genomic elements that show high levels of variation and therefore are useful tools for studying genetic polymorphism and constructing genetic linkage maps of eukaryotic organisms. Porphyra yezoensis Ueda is an economically important seaweed that is being targeted for genetic improvement using marker-assisted breeding. Hence, in an attempt to develop microsatellite markers for P. yezoensis, a microsatellite (or simple sequence repeat)-enriched library was constructed to identify (GA)n and (CA)n motifs. A total of 71 perfect microsatellite clones were identified, of which 30 simple sequence repeat primer pairs were developed. Of these, 24 (80%) amplified polymerase chain reaction products of expected sizes. Twelve primer pairs amplified two to four bands, whereas another 12 primer pairs produced monomorphic banding patterns. Data for 12 loci were analyzed using POPGENE software version 1.32. A total of 29 alleles were produced at 12 loci, with an average of 2.42 alleles (Na) and 1.81 effective alleles (Ne) per locus. These markers were used to analyze the genetic diversity within 11 geographically different lines of P. yezoensis. Overall, these lines were clustered into two divisions with those from close geographic locations clustering together. Further cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of motif repeats in different alleles were major sources of polymorphisms.     

Genetic analysis of the position of meiosis in <Emphasis Type="Italic">Porphyra haitanensis</Emphasis> Chang et Zheng (Bangiales,Rhodophyta)

Crossing experiments were carried out between artificial pigmentation mutants and the wild type in Porphyra haitanensis Chang et Zheng to ascertain where meiosis occurs in its life history by confirming whether the color segregation and the color-sectored blades appear in F₁ gametophytic blades developed from conchospores which are released from heterozygous conchocelis. Two red-type pigmentation mutants (R-10 and SPY-1) were used as the female parent. Their blades are red or red orange in color, thinner than the wild type and weak in elasticity, and have no denticles on their margins. The wild type (W) was used as the male parent; its blades are light brown in color, thick and good in elasticity, and have many marginal denticles. The F₁ gametophytic blades developed from conchospores which were released from heterozygous conchocelis produced in the crosses of R-10(♀)×W(♂) and SPY-1(♀)×W(♂) showed two parental colors (R and W) and two new colors (R', lighter in color than R; W', wild-type-like color and redder than W). Linear segregation of colors occurred in the F₁ blades, forming color-sectored blades with 2–4 sectors. In the color-sectored blades, R and R' sectors were thinner than W and W' sectors, and had weak elasticity and no denticles on their margins, whereas W and W' sectors were thick and had good elasticity and many marginal denticles. Of the F₁ gametophytic blades, 95.2–96.7% were color-sectored and only 3.3–4.8% were unsectored. These results indicate that meiosis of P. haitanensis occurs during the first two cell divisions of a germinating conchospore, and thus it is considered that the initial four cells of a developing conchosporeling constitute a linear genetic tetrad leading to the formation of a color-sectored blade. The new colors of R' and W' were recombinant colors due to the chromosome recombination during the first cell division in meiosis. It is considered that color phenotypes of the two mutants used in this paper were result of two (or more) recessive mutations in different genes, and that they also have mutations concerned with blade thickness and formation of marginal denticles, which are linked with the color mutations.     

Purification of phycoerythrin from <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Ueda (Bangiales,Rhodophyta) using expanded bed absorption

R-phycoerythrin was purified by means of phenyl-sepharose expanded bed absorption and DEAE-sepharose ion-exchange chromatography from Porphyra yezoensis, one of the largest and important aquaculture species in China. Final R-phycoerythrin preparation was characterized by purity ratio above 4 and the homogeneity in native PAGE, respectively. The results of absorption spectrum, fluorescence spectrum and SDS-PAGE were in agreement with previous reports on R-PE. The yield of R-phycoerythrin was 0.82 mg g⁻¹ wet leafy gametophyte of P. yezoensis. This method is a high-protein recovery technology while reducing processing time, and is suitable for the large batch production of R-phycoerythrin, which will enhance the value of P. yezoensis in China, especially the inferior P. yezoensis which can not be used for flake processing.     

Selection and characterization of a high-temperature tolerant strain of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis> Chang et Zheng (Bangiales,Rhodophyta)

Porphyra haitanensis is one of the most economical nutritive marine algae; however, its production and quality are significantly jeopardized by high temperatures. Selection of heat-resistant strains will greatly reduce the economic risks and benefit to the nori industry. Three previously isolated and improved strains with a high yield were screened at 28°C and identified, of which one strain, ZS-1, showed significantly improved heat tolerance. Upon further characterizing of the cultures of the ZS-1 strain and the wild-type (WT) strain at 28°C and 30°C, the ZS-1 conchospore germlings survived at rates of 69.9% and 59.6%, while the WT conchospore germlings survived at significantly lower rates of 15.9% and 6.7%, respectively, over a period of 15 days. Furthermore, ZS-1 conchospore germlings divided at significantly higher rates of 100% and 88.6% compared to the WT conchospore germlings with 90.4% and 63.8%, respectively. When the 35-day-old conchospore germlings were transferred from the optimal temperature of 24°C to higher temperatures of 28°C and 30°C, the ZS-1 blades sustained growth over a 25-day period without decay and increase of blade lengths with a factor of 18.5 and 10.3 times, respectively. The blade lengths of the WT germlings only increased by a factor of 1.7 and 0.9 times and began to decay after being cultured for 15 days at 28°C and 30°C. At 24, 28, and 30°C, the ZS-1 blades grew 3.4, 8.6, and 8.0 times faster than those of the WT. Evidently, ZS-1 is a fast-growing and heat-resistant strain compared to the WT strain and may offer an alternative for the nori industry.     

Interaction of photoperiod and temperature in the development of conchocelis of <Emphasis Type="Italic">Porphyra purpurea</Emphasis> (Rhodophyta: Bangiales)

The indoor cultivation of the free-living conchocelis of a Porphyra purpurea (Roth) C. Ag. strain, isolated from Long Island Sound, was established, and the effects of both photoperiod and cultural temperature on conchosporangia development were studied. Statistical analysis revealed that temperatures between 10°C and 15°C and light phases between 12 and 16 h per day comprised an ideal growth “window” for both the vegetative growth and reproductive development of conchocelis. For vegetative growth, there was a significant interaction between temperature and photoperiod. Conchospores were released from mature conchosporangia under both neutral (12/12 h) and long (16/8 h) day lengths. Different seawater supplements, such as full- and half-strength Von Stosch enrichment, showed no significantly different effects on growth or development. This work provides a guideline for maintaining conchocelis cultures of P. purpurea, which is a type of the Porphyra genus.     

Construction and characterization of a bacterial artificial chromosome library of marine macroalga<Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta)

A large-DNA-fragment library is necessary for research into thePorphyra genome. In this study, a bacterial artificial chromosome (BAC) library ofPorphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research inP. yezoensis. These authors contributed equally to this work.     

Isolation,Antimicrobial Activity,and Metabolites of Fungus <Emphasis Type="Italic">Cladosporium</Emphasis> sp. Associated with Red Alga <Emphasis Type="Italic">Porphyra yezoensis</Emphasis>

Cladosporium sp. isolate N5 was isolated as a dominant fungus from the healthy conchocelis of Porphyra yezoensis. In the re-infection test, it did not cause any pathogenic symptoms in the alga. Twenty-one cultural conditions were chosen to test its antimicrobial activity in order to obtain the best condition for large-scale fermentation. Phenylacetic acid, p-hydroxyphenylethyl alcohol, and L-beta-phenyllactic acid were isolated from the crude extract as strong antimicrobial compounds and they are the first reported secondary metabolites for the genus Cladosporium. In addition, the Cladosporium sp. produced the reported Porphyra yezoensis growth regulators phenylacetic acid and p-hydroxyphenylacetic acid. No cytotoxicity was found in the brine shrimp lethality test, which indicated that the environmental-friendly Cladosporium sp. could be used as a potential biocontrol agent to protect the alga from pathogens.     

Mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 and plastid <Emphasis Type="Italic">rbc</Emphasis>L genes of <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis> (Gracilariaceae,Rhodophyta)

The mitochondrial cytochrome c oxidase subunit I gene sequence was recently developed for DNA barcoding of red algal species. We determined the 1245 base pairs of the gene from 27 taxa of an agar-producing species, Gracilaria vermiculophylla, and putative relatives and compared the results with rbcL data from the same species. A total of 392 positions (31.5%) were variable, 282 positions (22.6%) were parsimoniously informative, and average sequence divergence was 13% in an ingroup. Within G. vermiculophylla, pairwise divergence of the gene was variable up to 11 bp (0.9%). Seven recognized haplotypes of cox1 tended to be geographically related. In the aligned 1386 bp of rbcL, three haplotypes were recognized. These results suggest that cox1 is a valuable molecular marker within species and will be very useful in haplotype analyses.     

Photosynthetic response of <Emphasis Type="Italic">Bangia fuscopurpurea</Emphasis> (Bangiales,Rhodophyta) towards dehydration and hyposalinity

Bangia fuscopurpurea, an important farmed species in China, inhabits upper intertidal zones where it suffers periodical desiccation and salinity stress. However, the physiological response and acclimation mechanism of Bangia to abiotic stress is unknown. Here, the photosynthetic response of B. fuscopurpurea to desiccation and hyposalinity was investigated by using chlorophyll fluorescence measurement. The optimum photosynthetic efficiency of photosystem II (Fv/Fm), photochemical quenching (qP) and the non-photochemical quenching (NPQ) of B. fuscopurpurea thalli maintained at basal level when the absolute water content (AWC) was 32%. As AWC decreased from 32% to 9%, Fv/Fm dropped from 0.62 to 0.1 and NPQ increased from 0.2 to 1.2. No significant change occurred in the mean qP but great standard deviation was present as AWC was 9%. Fv/Fm, qP and NPQ of the thalli with 9% AWC fully recovered after rehydration. That B. fuscopurpurea kept high photosystem II photochemical reactions even when AWC was mere 32% enabled this species to survive extreme air drying at low tide. Fv/Fm and qP dropped while NPQ increased with 1 h of varying hyposaline treatment and they regained the basal levels after 6–24 h treatment. Nine days later, Fv/Fm, qP and NPQ levels of the thalli in 100% freshwater was equal to the control level (0.62, 0.9, 0.1, respectively). The present finding suggested that this alga has high photosynthetic capacity to survive during low tide, even during heavy rainfall. We hope this study would facilitate further investigation on the stress acclimation mechanism of B. fuscopurpurea.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Simultaneous infection by red rot and chytrid diseases in <Emphasis Type="Italic">Porphyra yezoensis</Emphasis>Ueda

This paper presents the results of a study on the diseases of Porphyra yezoensisUeda along the north coast of China, where red rot (Pythium porphyrae) and the chytrid Olpidiopsis sp. diseases were both found to be present. Infection by the mycelia of Pythium porphyraeand the thallus of Olpidiopsis sp. was studied in detail. At the early stage of infecton the mycelia of Pythium porphyraeand the fungus of chytrid can be found in host cells at the same time. In the middle and late stages of the complication, it mainly appears as red rot disease, toward the end appearing almost completely as red rot disease. The complication even can be found on the cells of fronds from the freeze-storage nori nets. However, the freeze-storage nets can help prevent spread of the infection and improve nori quality.     

Polyamines stimulate non-photochemical quenching of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence in <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis>

Polyamines (PAs) are small metabolites that are produced and oxidized in chloroplasts with an obscure mode of action. Recently, we showed that qE is stimulated by PAs in higher plants (Nicotiana tabacum) and in genetically modified plants with elevated thylakoid-associated PAs (Ioannidis and Kotzabasis Biochim Biophys Acta 1767:1371–1382, 2007; Ioannidis et al. Biochim Biophys Acta 1787:1215–1222, 2009). Here, we investigated further their quenching properties both in vivo in green algae and in vitro is isolated LHCII. In vivo spermine up-regulates NPQ in Scenedesums obliquus about 30%. In vitro putrescine—the obligatory metabolic precursor of PAs—has a marginal quenching effect, while spermidine and spermine exhibit strong quenching abilities in isolated LHCII up to 40%. Based on available 3D models of LHCII we report a special cavity of about 600 Å³ and a near-by larger pocket in the trimeric LHCII that could be of importance for the stimulation of qE by amines.     

Morphological and photosynthetic variations in the process of spermatia formation from vegetative cells in <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Ueda (Bangiales,Rhodophyta) and their responses to desiccation

Porphyra yezoensis has a macroscopic foliage gametophyte phase with only a single cell layer, and is ideally suited for the study of the sexual differentiation process, from the vegetative cell to the spermatia. Firstly, we compared variations in the responses of the vegetative and male sectors to desiccation. Later, cell tracking experiments were carried out during the formation of spermatia from vegetative cells. The two sectors showed similar tolerance to desiccation, and the formation of spermatia from vegetative cells was independent of the degree of desiccation. Both light and scanning electron microscopy (SEM) observations of the differentiation process showed that the formation of spermatia could be divided into six phases: the one-cell, two-cell, four-cell, eight-cell, pre-release and spermatia phases. Photomicrographs of Fluorescent Brightener staining showed that the released spermatia had no cell walls. Photosynthetic data showed that there was a significant rise in Y(II) in the four-cell phase, indicating an increase in photosynthetic efficiency of PSII during this phase. We propose that this photosynthetic rise may be substantial and provide the increased energy needed for the formation and release of spermatia in P. yezoensis.