AXENIC TISSUE CULTURE AND MORPHOGENESIS IN PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)

To better understand developmental phenomena in macroalgal tissue culture, we examined the morphogenesis of Porphyra yezoensis Ueda (strain TU-1) cultured aseptically in defined synthetic media . Generally, the filamentous thalli (sporophyte; conchocelis phase) of P. yezoensis were densely tufted with uniseriate filaments. The foliose thalli (gametophyte) were monolayered. In this study, axenic filamentous thalli retained their characteristic morphogenesis; there were no obvious differences between morphogenetic traits in unialgal and axenic conditions. However, conchospores, which might have developed into the foliose form under unialgal conditions, germinated into calluslike masses under axenic conditions. Most of the gametophytes gradually lost their typical morphogenesis after the first longitudinal cell division. Some of the calluslike masses developed rhizoidlike structures in several places or along the entire mass. Therefore, we concluded that P. yezoensis, in axenic cultures, loses its typical morphogenesis only during the gametophytic phase. The axenic tissue culture of Porphyra established in this study is a promising assay system for the identification of growth and morphogenetic factors.     

VARIATIONS IN THE CELL WALLS AND PHOTOSYNTHETIC PROPERTIES OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA) DURING ARCHEOSPORE FORMATION1

The formation of archeospores is characteristic of Porphyra yezoensis Ueda and is important for Porphyra aquaculture. Recently, it has been regarded as a valuable seed source for propagation of thalli in mariculture. Cell wall composition changes are associated with archeospore formation in P. yezoensis. Here, we report changes of cell walls of P. yezoensis during archeospore formation. The surfaces of vegetative cells that were originally smooth became rougher and more protuberant as archeosporangia were formed. Ultimately, the cell walls of archeosporangia ruptured, and archeospores were released from the torn cell walls that were left at distal margins of thalli. With changes in cell walls, both effective quantum yield and maximal quantum yield of the same regions in thalli gradually increased during the transformation of vegetative cells to archeospores, suggesting that the photosynthetic properties of the same regions in thalli gradually increased. Meanwhile, photosynthetic parameters for different sectors of thalli were determined, which included the proximal vegetative cells, archeosporangia, and newly released archeospores. The changes in photosynthetic properties of different sectors of thalli were in accordance with that of the same regions in thalli at different stages. In addition, the photosynthetic responses of archeosporangia to light showed higher saturating irradiance levels than those of vegetative cells. All these results suggest that archeosporangial cell walls were not degraded prior to release but were ruptured via bulging of the archeospore within the sporangium, and ultimately, archeospores were discharged. The accumulation of carbohydrates during archeospore formation in P. yezoensis might be required for the release of archeospores.     

ALLOPOLYPLOIDY IN NATURAL AND CULTIVATED POPULATIONS OF PORPHYRA (BANGIALES,RHODOPHYTA)1

To confirm whether allopolyploidy occurs in samples of previously identified Porphyra yezoensis Ueda, P. tenera Kjellm., and P. yezoensis × P. tenera from natural and cultivated populations, we examined these samples by using PCR‐RFLP and microsatellite analyses of multiple nuclear and chloroplast regions [nuclear regions: type II DNA topoisomerase gene (TOP2), actin‐related protein 4 gene (ARP4), internal transcribed spacer (ITS) rDNA and three microsatellite loci; chloroplast region: RUBISCO spacer]. Except for the ITS region, these multiple nuclear markers indicated that the wild strain MT‐1 and the cultivated strain 90‐02 (previously identified as P. yezoensis × P. tenera and cultivated P. tenera, respectively) are heterozygous and possess both genotypes of P. tenera and P. yezoensis in the conchocelis phase. Furthermore, gametophytic blades of two pure lines, HG‐TY1 and HG‐TY2 (F₁ strains of MT‐1 and 90‐02, respectively), were also heterozygous, and six chromosomes per single cell could be observed in each blade of the two pure lines. These results demonstrate that allopolyploidy occurs in Porphyra strains derived from both natural and cultivated populations, even though ITS genotypes of these strains showed homogenization toward one parental ITS.     

TWO SPECIFIC CAUSES OF CELL MORTALITY IN FREEZE‐THAW CYCLE OF YOUNG THALLI OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Porphyra yezoensis Ueda is an important marine aquaculture crop with single‐layered gametophytic thalli. In this work, the influences of thallus dehydration level, cold‐preservation (freezing) time, and thawing temperature on the photosynthetic recovery of young P. yezoensis thalli were investigated employing an imaging pulse‐amplitude‐modulation (PAM) fluorometer. The results showed that after 40 d of frozen storage when performing thallus thawing under 10°C, the water content of the thalli showed obvious effects on the photosynthetic recovery of the frozen thalli. The thalli with absolute water content (AWC) of 10%–40% manifested obvious superiority compared to the thalli with other AWCs, while the thalli thawed at 20°C showed very high survival rate (93.10%) and no obvious correlation between thallus AWCs and thallus viabilities. These results indicated that inappropriate thallus water content contributed to the cell damage during the freeze‐thaw cycle and that proper thawing temperature is very crucial. Therefore, AWC between 10% and 40% is the suitable thallus water content range for frozen storage, and the thawing process should be as short as possible. However, it is also shown that for short‐term cold storage the Porphyra thallus water content also showed no obvious effect on the photosynthetic recovery of the thalli, and the survival rate was extremely high (100%). These results indicated that freezing time is also a paramount contributor of the cell damage during the freeze‐thaw cycle. Therefore, the frozen nets should be used as soon as time permits.     

KARYOLOGY OF BANGIA ATROPURPUREA (RHODOPHYTA,BANGIALES) FROM MEDITERRANEAN AND NORTHEASTERN ATLANTIC POPULATIONS1

We studied the karyology of Bangia atropurpurea (Roth) C. Ag. collected from marine and freshwater populations from the Mediterranean region and some northeastern Atlantic localities. Gametophytic thalli had two haploid karyotypes, n = 3 and n = 4. The n = 4 karyotype was only occasionally present in the Mediterranean and was also found in one Atlantic population, confirming a previous report. We propose that the four-chromosome karyotype is an aneuploid form, n + 1. Chromosomes were frequently observed either in a parallel arrangement or in a circular configuration.     

MORPHOLOGICAL AND MOLECULAR ANALYSIS OF THE ENDANGERED SPECIES PORPHYRA TENERA (BANGIALES,RHODOPHYTA)1

Porphyra tenera Kjellman, widely cultivated in nori farms before the development of artificial seeding, is currently listed as an endangered species in Japan. To confirm whether a wild‐collected gametophytic blade was P. tenera or the closely related species P. yezoensis Ueda, morphological observations and molecular analyses were made on the pure line HGT‐1 isolated from a wild blade. This pure line was identified as P. tenera based on detailed morphological features. Sequences of the nuclear internal transcribed spacer region 1 and the plastid RUBISCO spacer revealed that P. tenera HGT‐1 was clearly different from P. yezoensis f. narawaensis Miura, the main species cultivated in Japan. PCR‐RFLP analysis of the internal transcribed spacer region was found to be a convenient method for rapid discrimination between P. tenera and cultivated P. yezoensis. The restriction patterns generated by the endonucleases Dra I and Hae III were useful for differentiating between both gametophytic and conchocelis stages of P. tenera HGT‐1 and P. yezoensis f. narawaensis strains. Thus, PCR‐RFLP analysis will serve as a valuable tool for rapid species identification of cultivated Porphyra strains, culture collections of Porphyra strains for breeding material and conservation of biodiversity, and, as codominant cleaved amplified polymorphic sequence markers for interspecific hybridization products between P. tenera and P. yezoensis f. narawaensis. Under the same culture conditions, rate of blade length increase and the blade length‐to‐width ratio were lower in P. tenera HGT‐1 than in P. yezoensis f. narawaensis HG‐4. The HGT‐1 became mature more rapidly than HG‐4 and had thinner blades.     

ULTRASTRUCTURE OF GERMINATING CARPOSPORES OF PORPHYRA VARIEGATA (KJELLM.) HUS (BANGIALES,RHODOPHYTA)1

The fine structure of released, attached, and germinating carpospores of Porphyra variegata (Kjellm.) Hus is described. Adhesive vesicles, formed during sporogenesis and discharged upon settling of the spore, produced a layer of adhesive mucilage around the spore and filled a deep imagination on the spore's ventral side. The mucilage layer was punctured by the emergence of a germ tube. Both spore and germ tube were lined by newly deposited cell wall. Germination was accompanied by vacuolation and starch mobilization. The morphological development of the sporeling was not noticeably influenced by the great variability of the timing, location, and orientation of septum formation. The attached carpospore possessed a plastid like that of gametophyte cells: stellate with one large central pyrenoid and no peripheral encircling thylakoids. Cells of mature vegetative cells of the conchocelis had plastids that were elongate and parietal and had multiple pyrenoids and encircling thylakoids. Most stages in the transition between the two forms of plastids occurred during carpospore germination.     

A GAMETOPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) CONTAINS FOUR APPARENT POLYSACCHARIDE-BINDING DOMAINS1

A complementary DNA(cDNA)clone from a Porphyra purpurea (Roth) C. Agardh gametophyte-specific subtracted cDNA library was found to encode a protein containing a signal peptide and four very similar regions with a high degree of amino acid sequence similarity to the cellulose-binding domains of fungal celluloses. Northern hybridization analysis indicated that the messenger RNA of this cDNA is highly abundant in the gametophyte but not detectable in the sporophyte. In vitro translation of the cDNA in the presence of canine pancreatic microsomes demonstrated that the signal peptide is capable of directing the protein into the endoplasmic reticulum where it is glycosylated. Because these observations suggested a possible role as a gametophyte-specific cell wall protein, cell wall protein, were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this was the protein encoded by the cDNA. The abundance and organization of this protein suggest a role as a cell wall structural protein involved in cross-linking polysaccharides.     

ASSESSMENT OF PHOTOSYNTHETIC PERFORMANCE OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA) IN CONCHOCELIS PHASE1

Photosynthetic characteristics of four Porphyra yezoensis Ueda [a taxonomic synonym of Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi] strains in conchocelis phase were investigated and compared with one wildtype of P. yezoensis and two strains of Porphyra haitanensis T. J. Chang et B. F. Zheng [a taxonomic synonym of Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata]. Results showed that experimental strains had higher contents of chl a and carotenoids, but a lower content of total phycobiliproteins than the wildtype. Meanwhile, photochemical efficiency of PSII was measured using pulse amplitude modulation (PAM) fluorometry technology. The value of PSII photosynthetic parameters of P. yezoensis strains were all higher than the wild strain, and the maximal quantum yields (F_v/F_m), effective quantum yields Y(II), and relative photosynthetic electron transport rates (rETR) of P. haitanensis were higher than those of P. yezoensis. The present study verified the possibility of selective breeding of P. yezoensis using the filamentous sporophyte instead of the gametophytic thallus, the advantages being (i) nonrequirement of control of life cycle and (ii) direct and rapid cultivar improvement by artificial selection. We consider the method to be a promising technique for selective breeding of P. yezoensis cultivars.     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

MEIOSIS,BLADE DEVELOPMENT,AND SEX DETERMINATION IN PORPHYRA PURPUREA (RHODOPHYTA)1

The discovery in the early 1980s that meiosis occurs during germination of conchospores of Porphyra yezoensis Ueda suggested that the sexually divided fronds of Porphyra purpurea (Roth) C. Agardh might similarly originate from meiotic segregation of a pair of sex-determining alleles during early sporeling development. After establishing conditions suitable for propagating P. purpurea in culture, observations on developing sporelings demonstrated that meiosis takes place during the first two divisions of the germinating conchospores. In the first division, the spore is split into an upper and lower cell. In the second, an anticlinal division in the upper cell yields two daughter cells situated one beside the other, and a periclinal division in the bottom cell gives two cells arranged one above the other. Thus, during normal development, the first four cells of the sporeling constitute a meiotic tetrad whose cells are arranged in a characteristic fashion. Stable color mutants of P. purpurea were isolated, genetically characterized, and used as genetic markers to follow the fate of individual cells of the tetrad during subsequent frond development. Nearly the entire blade of the mature thallus is derived from the two upper cells of the tetrad, with the two lower cells mostly giving rise to the rhizoidal holdfast region. Cell lineage boundaries laid down by the segregation of color alleles at meiosis corresponded perfectly with those later defined by sexual differentiation on the same fronds, strongly supporting the hypothesis that sex determination in P. purpurea is controlled by alleles at a segregating chromosomal locus.     

IDENTIFICATION OF PORPHYRA HAITANENSIS (BANGIALES,RHODOPHYTA) MEIOSIS BY SIMPLE SEQUENCE REPEAT MARKERS1

The simple sequence repeat (SSR) marks were employed to identify the stage at which meiosis occurs in the life cycle of Porphyra haitanensis T. J. Chang et B. F. Zheng. More than 90% of F1 blades of heterozygous conchocelis produced by the cross between a red mutant (R, ♀) and the wildtype (W, ♂) were color sectored. Two parental colors (R and W) and two new colors (R′ and W′) appeared in linear sectors in the color‐sectored F1 blades. Two SSR primer pairs selected from a total of 52 primer pairs generated a specific paternal and maternal fragment, respectively. Co‐occurrence of these two bands was detected in heterozygous conchocelis and in the color‐sectored F1 blades with two to four sectors, such as R + W, R′ + W′, and R′ + R + W + W′. However, the single‐colored F1 blades exhibited only one band. In the sectors isolated from the color‐sectored F1 blades, R and R′ were the same, showing the maternal pattern, whereas W and W′ were the same, showing the paternal pattern. These data suggested that the two different bands from heterozygous conchocelis originated from the parents and segregated in the F1 blades, whereas the two new colors, R′ and W′, in the F1 blades were produced by the exchange and recombination of alleles of the parental colors during meiosis. These results indicated that meiosis of P. haitanensis occurs during the first two cell divisions of a germinating conchospore, and, therefore, the initial four cells constitute a linear genetic tetrad, leading to the formation of a color‐sectored blade.     

GROWTH AND MORPHOLOGY OF YOUNG GAMETOPHYTES OF PORPHYRA ABBOTTAE (RHODOPHYTA): EFFECTS OF ENVIRONMENTAL FACTORS IN CULTURE1

Growth, blade shape and blade thickness of young gametophytes of Porphyra abbottae Krishnamurthy cultured from conchospores were determined at various combinations of temperature (8, 10, 12° C), photon flux density (17.5, 70, 140 μmol·m-^?2·S^?1), nutrient concentration (5, 25, 50, 100% f medium) and water motion (0, 50, 100, 150 rpm). Growth (as surface area) was light-saturated at 70 μmol· m^?2· S^?1, light-inhabited at 140 μmol·m^?2· S^?1, and nutrient-saturated an 25% f medium. Temperature had no significant effect on growth. Water motion and nutrients had an interactive effect on growth, with water motion having the greatest effect at the lowest nutrient concentrations. Water motion enhanced growth even at saturating nutrient concentrations. Blade length / width ratio was greater in low light (2.5) than in saturating light (1.9); with increasing water motion the ratio increased from 1.2 to 2.4. Blade thickness (53-88 μm) was greatest at the highest nutrient concentrations and at the lowest water motion levels. Temperature and light did not have a consistent effect on blade thickness.     

IDENTIFICATION OF CYTOKININS IN SARGASSUM MUTICUM (PHAEOPHYTA) AND PORPHYRA PERFORATA (RHODOPHYTA)1

Combined gas chromatography-mass spectrometry (GCMS) was used to identify and quantify specific cytokinins from Porphyra perforate J. Ag. and Sargassum muticum (Yendo) Fensh. The level of isopentenyladenosine was estimated to be 0.6 μ·kg^?1 fresh weight in Porphyra and 0.9 μ·kg^?1 fresh weight in Sargassum. The level of cis-zeatin riboside was estimated to be 0.2 μ·kg^?1 fresh weight in Sargassum. This is the first definitive identification of a cytokinin from a red alga, and the second report from a brown alga.     

IDENTIFICATION OF CROSS‐FERTILIZED CONCHOCELIS USING CLEAVED AMPLIFIED POLYMORPHIC SEQUENCE MARKERS IN CROSS‐EXPERIMENTS OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

As a part of the construction of a Porphyra yezoensis Ueda genetic linkage map, we conducted intraspecific cross‐experiments and subsequent screening of cross‐fertilized conchocelis by cleaved amplified polymorphic sequence (CAPS) analysis. The cross‐experiments were carried out between males of the wildtype (KGJ) and females of the recessive green mutant (TU‐2) using two methods, controlled and random crosses. A total of 42 and 186 wildtype‐colored conchocelis colonies were obtained from the former and latter experiments, respectively. Among those, 49 DNA samples (14% and 23% obtained from the former and latter crosses, respectively) showed biparental CAPS patterns in the two gene regions (EF‐1α open reading frame [ORF] region and V‐ATPase). This study represents the first report in which the cross‐fertilized conchocelis of P. yezoensis has been directly confirmed by molecular marker. The combination of the simple DNA extraction and CAPS analysis may be applicable in genetic studies of other macroalgae that are monoecious and/or grow slowly in laboratory culture.     

LITHIUM CHLORIDE EXTRACTION OF DNA FROM THE SEAWEED PORPHYRA PERFORATA (RHODOPHYTA)1

Lithium chloride facilitates the softening of cell walls resulting in a simple, quick (2 h) method for DNA extraction from the red seaweed Porphyra perforata J. Agardh. A 5-min treatment of tissues in Lid at 55°C extracts DNA that is relatively free of the viscous polysaccharides and proteins that are usually coextracted in large amounts from cell walls and cytoplasm. This protocol does not require grinding of tissues, hydroxyapatite binding, cetyl trimethyl ammonium bromide treatments, enzymatic treatments, phenol extraction, or CsCl-gradient centrifugation. The resulting DNA is of sufficient quality to be used as a template for polymerase chain reaction amplification.     

PROTOPLAST ISOLATION AND CELL DIVISION IN THE AGAR-PRODUCING SEAWEED GRACILARIA (RHODOPHYTA)1

Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber-van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid-producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R-10, 3% Macerozyme R-10, 1% agarase and 0.5% Pectolyase Y- 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2-6 days; cell division in 5-10 days. Protoplast-produced cell masses up to the 16-32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.