The physiological links of the increased photosystem II activity in moderately desiccated Porphyra haitanensis (Bangiales, Rhodophyta) to the cyclic electron flow during desiccation and re-hydration

Photosynthetic electron flow changed considerably during desiccation and re-hydration of the intertidal macroalgae Porphyra haitanensis. Activities of both photosystem (PSI) and photosystem (PSII) increased significantly at moderate desiccation levels. Whereas PSII activity was abolished at an absolute water content (AWC) <24 %, PSI remained active with progressive decreases in AWC to values as low as 16 %. This result suggested that cyclic electron flow around PSI was still active after inactivation of linear electron flow following severe desiccation. Moreover, the PSI activity was restored more rapidly than that of PSII upon re-hydration. Pretreatment of the blades with 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) suppressed PSII activity following desiccation to an AWC of ~16 % AWC. Cyclic electron flow around PSI decreased markedly in blades pretreated with DCMU than in blades without pretreatment of DCMU during re-hydration in seawater containing DCMU. All results suggested that the activity of PSII under desiccation conditions plays an important role in the operation of cyclic electron flow during desiccation and its recovery during re-hydration. Therefore, we proposed the PSII activity during desiccation could eventually lead to the accumulation of NADPH, which could serve as electron donor for P700⁺ and promote its recovery during re-hydration, thereby favoring the operation of cyclic electron flow.     

Estimation of the degree of self-fertilization in Porphyra yezoensis (Bangiales,Rhodophyta)

Crosses between genotypically distinct thalli of the monoecious species Porphyra yezoensis were carried out using immature thallus fragments from green- and red-type color mutants and also wild-type thalli. As the genes governing the mutants are monogenic, recessive to the wild-type, and belong to the same linkage group, the degree of self-fertilization could be estimated based on the pigmentation of the resultant diploid conchocelis. The degree of self-fertilization in the cross between the green-type and the wild-type was 48.5–55.0%, and in the cross between the red-type and the wild-type was 45.1–56.5%. In the cross between the green- and red-type mutants, the degree of self-fertilization was 46.0–54.5% when the green-type was the female parent, and was 44.8–55.6% when the red-type was the female parent.     

Rapid extraction of high-quality genomic DNA from Porphyra yezoensis (Bangiales, Rhodophyta)

We developed a simple, rapid and stable method for extraction of high molecular weight DNA from the marine red alga Porphyra yezoensis Ueda using both guanidium treatment and QIAGEN? kit (Funakoshi, Tokyo, Japan). The method does not require expensive equipment and complex steps. The DNA yield averaged 1.5 μg 100 mg^?1 of Porphyra tissue and the A260/A280 and A230/A260 ratios of the DNA were approximately 1.8 and 0.4, respectively. It was of sufficient quality to be used for not only polymerase chain reactions but also other DNA manipulation techniques such as restriction digestion and construction of genomic libraries.     

Induction and characterization of pigmentation mutants in Porphyra yezoensis (Bangiales, Rhodophyta)

Porphyra yezoensis Ueda conchospore germlings (1–4-cell stages) were treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) for inducing mutations. Three kinds of color-mutated gametophytic blades, which were composed of the mutated cells wholly, sectorially or spottedly, were obtained; and most of them were sectorially variegated blades. The highest frequency of these mutated blades was 1.3%. Four different pigmentation mutant strains were obtained by regenerating single cells and protoplasts that were enzymatically isolated from the mutated sectors of the sectorially variegated blades. The mutants were relatively stable in color in both gametophytic blade and conchocelis phases. In the two phases, each mutant strain showed characteristic differences in the in vivo absorption spectra, and had different pigment contents of major photosynthetic pigments (chlorophyll a, phycoerythrin and phycocyanin) as compared with the wild-type and with each other. The gametophytic blades from the four mutant lines showed significant differences in growth and photosynthetic rates, when they were cultured in the same conditions. By crossing the mutant with the wild-type, it was found that the color phenotypes of two mutants reported above, were resulted from two mutations in different genes, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic analysis of artificial pigmentation mutants in Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

Porphyra yezoensis Ueda artificial pigmentation mutants, yel (green), fre (red‐orange) and bop (pink), obtained by treatment with /V‐methyl‐/V′‐nitro‐N‐nitrosoguanidine, were genetically analysed. The mutations associated with color phenotypes are recessive because all of the heterozygous conchocelis resembled the wild type color when they were crossed with the wild type (wt). In the reciprocal crosses of yel × wt, both parental colors and eight types of blades appeared in the F₁ gametophytic blades from the heterozygous conchocelis. Both colors segregated in the sectored F₁ blades in a 1:1 ratio, indicating that the color pheno‐type of yel resulted from a single mutation in the nuclear gene. In the reciprocal crosses of fre × wt, however, four colors and more than 40 types of blades appeared in the F₁ blades from the heterozygous conchocelis, indicating that the color phenotype of fre resulted from two mutations in different genes. In the reciprocal crosses of bop×wt, three colors and 12 types of blades were observed in the F₁ blades from the heterozygous conchocelis. Both parental colors appeared far more frequently than the third new color. These results indicated that the color phenotype of bop resulted from two closely linked mutations in different genes, and the epistasis occurred in the F₁ blades. The mutants, yel, fre and bop, differ from the spontaneous green (C‐O), the red (H‐25) and the violet (V‐O) mutants of P. yezoensis, respectively.     

Morphological and AFLP variation of Porphyra yezoensis Ueda form, narawaensis Miura (Bangiales, Rhodophyta)

Detailed morphological observations were made on two strains of cultivated Porphyra: HG‐1 (pure line isolated from Dai‐1) and Noriken‐4 (parental strain of a pure line HG‐4). The two strains were identified as P. yezoensis f. narawaensis based on their macroscopic and microscopic features, such as long linear or oblanceolate blades up to 50 cm in maximum length, division formulae of spermatangia and zygotosporangia, shape of trichogynes and carpogonia, and the second transverse divisional plane formed at the division from c/2 to c/4 in zygotosporangia. Gametophytic blades from two completely homozygous conchocelis strains isolated in this study (HG‐1 and HG‐4) were cultured under the same conditions and compared to confirm whether the differences in their shapes are genetically determined. The shape of blades from both of conchospores and monospores was always more slender in HG‐4 than in HG‐1 at the same blade age, suggesting that the difference in the blade shape between the two pure lines is due to genetic variation. To estimate the level of genetic variation the two pure lines were subjected to amplified fragment length polymorphism fingerprint analysis. A total of 230 bands were detected in HG‐1 and HG‐4 using eight selective primer pairs, and the number of polymorphic bands was only two in HG‐1. These results indicate that the two pure lines certainly show genetic variation, which is, however, at an extremely low level. The importance of pure‐line breeding and the origin of currently cultivated Porphyra are discussed. This is the first report to identify currently cultivated Porphyra strains in Japan based on combined results of detailed morphological observations and molecular analysis.     

Evaluation of an improved strain of Porphyra yezoensis Ueda (Bangiales, Rhodophyta) with high-temperature tolerance

The red algae Porphyra yezoensis has high commercial and nutritional value; however, its cultivation and product quality are jeopardized by global warming. Screening of high-temperature-tolerant strains would greatly reduce cultivation risks and benefit the Porphyra industry. Single somatic cells isolated enzymatically from the wild-type (WT) blades irradiated by ⁶⁰Co-?? rays were screened at 25°C; one strain, T-17, displayed significant high-temperature resistance. Further culture studies showed that conchospore germlings of T-17 showed 76.2% survival at 23°C and 65.7% survival at 24°C, while the WT conchospore germlings survived at significantly lower percentages of 16.9% and 11.5%, respectively, over a period of 16 days. Furthermore, T-17 conchospore germlings divided at higher percentages of 100% at both 23 and 24°C compared to the WT conchospores with 99.8% and 81.8%, respectively. When the 50-day-old F₁ gametophytic blades were transferred from the optimal temperature of 18°C to high temperatures of 23 and 24°C, the T-17 blades sustained growth over a 45-day period without rot, and their mean lengths increased by 20 and 4.2 times, respectively. The mean lengths of WT blades only increased by a factor of 0.6 and 0.4 times and were severely decayed after being cultured for 15 days at high temperatures. The mean wet weights of T-17 blades increased by 418.9 and 195.3 times, but only 7.8 and 4.6 times in WT at 23 and 24°C, respectively. These results indicate that T-17 is superior to WT in growth and high-temperature tolerance, which may offer a new cultivar for the nori industry as a high-temperature-resistant strain to counteract the effects of global warming.     

Nuclear division of the vegetative cells, conchosporangial cells and conchospores of Porphyra yezoensis (Bangiales, Rhodophyta)

To confirm the position and timing of meiosis in Porphyra yezoensis Ueda, the nuclear division of vegetative cells, conchosporangial cells and conchospores was observed. An improved staining method using modified carbol fuchsin was introduced to stain the chromosomes of Porphyra. Pit‐connections between conchosporangial cells also stained well with this method. Leptotene, zygotene, pachytene, diplotene, diakinesis, metaphase, anaphase and telophase were observed in the conchosporangial cells. During the germination of conchospores, no characteristics of meiosis I were found. No difference between the nuclear division of vegetative cells and that of conchospores was observed, and 2–3 days were needed for the first cell division both in vegetative cells and conchospores. Therefore, the cell division that occurs during conchospore germination is not meiosis I. Our results indicate that the prophase of meiosis I begins during the formation of conchosporangial branches, and metaphase I, anaphase I and telophase I take place during the maturation of conchosporangial branches. Then the three‐bivalent nucleate sporangia complete cell division to form two individual conchospores, each with one three‐univalent nucleus. The conchospores released from the sporangia are at meiotic interphase. Meiosis II occurs at the first nuclear division during conchospore germination, which is a possible explanation for the observation of mosaic thalli in mutant germlings of P. yezoensis. The mosaic thalli might also arise from gene conversion/post meiotic segregation events, comparable to those in Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. and Neurospora crassa Shear & B.O. Dodge.     

Induction and isolation of pigmentation mutants of Porphyra yezoensis (Bangiales, Rhodophyta) by heavy-ion beam irradiation

The present study describes the isolation of pigmentation mutants of Porphyra yezoensis Ueda induced by heavy-ion beam irradiation for the first time. The gametophytic blades were irradiated with ¹²C⁺⁶ ion beams within a dose range of 25–400 Gy. From the survival rate and cell growth of the irradiated blades, it is suggested that a dose of 150 Gy or less is suitable to induce mutation for the isolation of mutants of P. yezoensis . After irradiation, red, green and deep reddish brown-colored gametophytic blades developed from archeospores that were released from each of the mutated cell clusters of the respective different colors, and the red mutant strain (IBY-R1) and green mutant strain (IBY-G1) were established as a conchocelis colony in culture. Blades of the mutants were characterized by their growth and photosynthetic pigment contents compared with those of the wild-type. From these results, it is clear that heavy-ion beam mutagenesis will be an effective tool for genetic and breeding studies of Porphyra , and also for other algal research.     

Research Note: Simple molecular discrimination of cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and related wild species (Bangiales,Rhodophyta)

To discriminate between cultivated Porphyra species (Porphyra yezoensis and Porphyra tenera) and closely related wild Porphyra species, we developed a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis of the rbcL gene using five restriction enzymes. Although our previous PCR‐RFLP analyses of internal transcribed spacer (ITS) rDNA and plastid RuBisCO spacer regions could not always discriminate wild P. yezoensis, wild P. tenera, and closely related wild species, the PCR‐RFLP profiles of the rbcL gene were useful in discriminating samples collected from natural habitats. Therefore, PCR‐RFLP analysis of the rbcL gene will help in the simple identification of a large number of samples, not only for the establishment of reliable cultures as breeding material, but also for the taxonomic investigations of species that are closely related to cultivated Porphyra.     

High monospore-producing mutants obtained by treatment with MNNG in Porphyra yezoensis Ueda (Bangiales,Rhodophyta)

Hydrobiologia - Two high monospore-producing pigmentation mutants were obtained by treatment with MNNG in Porphyra yezoensis Ueda. The mutants produced many monospores in young gametophytic blades...     

Characterizing the microbial culprit of white spot disease of the conchocelis stage of Porphyra yezoensis (Bangiales, Rhodophyta)

White spot disease is the most frequent and harmful disease affecting the shell-boring conchocelis stage of the economically important red alga Porphyra yezoensis (= Pyropia yezoensis (Ueda) Hwang & Choi) (Bangiales, Rhodophyta). To date, its potential pathogens and pathogenesis remain poorly understood. In this study, we isolated 7 bacteria, 26 fungi, and 10 actinomycetes from sick conchocelis. Re-infection experiments revealed that only one fungus, GF014, caused disease. Morphological characteristics of the colony, mycelium, sporangium, and spore of GF014 suggested that the strain belongs to the order Pleosporales and the genus Phoma. Classification based on internal transcribed spacer (ITS) sequences supported the results of morphological identification. These results indicate that GF014 is a species of Phoma and a specific pathogen for Porphyra. GF014 preferred organic nitrogen sources, and 20–28 °C and low salinity water were its optimal living conditions. Our findings will play an active role in preventing and treating white spot disease in P. yezoensis.     

Speciation in the marine crop Pyropia yezoensis (Bangiales,Rhodophyta)

In the marine red alga Pyropia yezoensis, commonly known in Japan as nori, sympatric occurrence of two cryptic species Pyropia sp. 2 and Pyropia sp. 3 on the same rock in a natural habitat has been confirmed by molecular analysis and detailed morphological observations. To confirm whether Pyropia sp. 2 and Pyropia sp. 3 were reproductively isolated in the sympatric population, 170 blades that had previously been studied using a maternally inherited plastid marker were examined with a nuclear gene marker. The results suggested that Pyropia sp. 2 and Pyropia sp. 3 with identical morphological features were reproductively isolated in the sympatric population and that they were different species based on the biological species concept. Although gametophytic blades of Pyropia were usually assumed to be haploid, 18 of 170 blades possessed both of the two genotypes derived from Pyropia sp. 2 and from Pyropia sp. 3. These results inferred that allodiploid blades were generated from the interspecific hybridization between these two cryptic species. The present findings provide insights for future studies on the speciation mechanism in seaweeds, particularly for genera that contain numerous species.     

Protoplast isolation and fusion in Porphyra (Bangiales,Rhodophyta)

Two sediment sampling campaigns were conducted in 1978 and 1988 in Lake Geneva (Switzerland). Organic carbon, total nitrogen, total phosphorus and its various forms were analyzed. Results indicate a stability of organic carbon and nitrogen mass, and a significant increase of phosphorus. The variation of phosphorus mass is related to the increase of nonapatite inorganic phosphorus. This study attempts to quantify the phosphorus exchanges at the water sediment interface. The dissolved oxygen level in the bottom water determines the exchange direction. In aerobic conditions, sediments accumulate the excess of phosphorus, while in anaerobic conditions, they constitute an internal source.     

Effects of light photon flux density and spectral quality on photosynthesis and respiration in Porphyra yezoensis (Bangiales, Rhodophyta)

A comparative study on the effects of photon flux density and spectral quality on photosynthesis and respiration in the marine red alga Porphyra yezoensis Ueda was conducted using a light dispenser. Results showed that the photosynthetic response, expressed in light utilization efficiency (LUE) for preselected wave bands of photosynthetically active radiation, could be ranked as follows: white > green > red > blue. Differences in LUE were also found between conchocelis and gametophyte stages and different strains of the alga. Pre-illumination light compensation, post-illumination light compensation, light saturation, respiration and photorespiration were also measured and compared. The relationships between light exposure, photosynthetic capacity, and the natural environmental conditions are discussed.     

Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta)

Nine primary regenerants were recovered by interspecific protoplast fusion of Porphyra yezoensis Ueda T‐14 (Py) (cultivated Porphyra) and Porphyra tenuipedalis Miura (Pt). This combination is difficult to achieve with conventional sexual hybridization, yet is important in that non‐cultivated P. tenuipedalis is partially resistant (PR) to red rot disease, caused by the microbial pathogen, Pythium porphyrae Takahashi et Sasaki. Out of the nine primary regenerants, two strains (Py‐Pt‐4 and Py‐Pt‐7) were like the parent, P. tenuipedalis, while the rest were like the other cultivated parent P. yezoensis T‐14 in their life cycle. Red rot resistance was assessed in parents and interspecific fusion product progeny (FPP) by exposing the foliose thalli to equivalent infection and measuring two parameters of the host‐pathogen interactions: supported fungal biomass and amount of disease produced. Intermediate resistance between P. yezoensis T‐14 (1.00) and P. tenuipedalis (0.13) was observed in two of the Py‐type FPP, Py‐Pt‐2F₂ (0.25) and Py‐Pt‐5F₂ (0.23). Stable inheritance of resistance was observed through two subsequent generations. The morphologic and reproductive characteristics of the regenerated foliose thalli, and nature of host‐pathogen interactions were used to further verify the hybrid origin of the FPP. Host‐pathogen interactions were followed using epi‐fluorescence and scanning electron microscopy (SEM). The zoospores encysted at higher rates on the susceptible cultivated parent (P. yezoensis T‐14) germinated immediately and the short germ tubes formed appres‐soria and penetrated the algal cells near the site of encystment. While on the PR parental (P. tenuipedalis) and partially resistant FPP (PRFPP) progeny (Py‐Pt‐2F₂ and Py‐Pt‐5F₂) the low rate of zoospore encystment was followed by cyst germination, but only a few of the germ tubes formed appressoria and penetrated the thallus surface. Long germ tubes (with no appressoria) were seen growing on the thallus surface without host penetration. The minimal rate of encystment concomitant with low rate of appressorium formation on the PR parent and PRFPP was observed as the major factor responsible for the partial resistance in these thalli.     

Rapid DNA extraction from conchocelis and ITS-1 rDNA sequences of seven strains of cultivated Porphyra yezoensis (Bangiales, Rhodophyta)

In order to extract DNA rapidly from cultivated Porphyra, we extracted total DNA from conchocelis using the ISOPLANT II kit (Nippon Gene) without liquid nitrogen treatment or CsCl-gradient ultracentrifugation. By confirming the reproducibility of RAPD patterns, it is concluded that the quality of the extracted DNA is sufficient to use as a template for molecular investigation. Using this rapid method, the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions was amplified from seven strains of cultivated Porphyra, which had been maintained as free-living conchocelis by subculturing in the laboratory. From the amplified DNAs, the ITS-1 sequences were determined in order to identify the species and genetic relationship of the strains. The sequences were identical in the seven strains, and all the strains were identified as P. yezoensis. Furthermore, the gametophytic blades of these strains showed long linear or oblanceolate shapes in the laboratory culture. It was concluded that these strains are P. yezoensis form. narawaensis. This rapid DNA extraction method from conchocelis will be a powerful tool for phylogenetic analysis and for genetic improvement of cultivated Porphyra.     

Complete mitochondrial genome sequence of Pyropia yezoensis (Bangiales,Rhodophyta) from Korea

Species of the genera Porphyra and Pyropia are the major seaweed cultivars in Korea, Japan, and China. Genomic data from Porphyra/Pyropia species is now being used for molecular breeding and bioengineering. We determined the complete mitochondrial genome (mtDNA) sequence of Pyropia yezoensis (Bangiales, Rhodophyta) from Korea. Pyropia yezoensis mtDNA was 35,596 bp in size and exhibited high sequence similarity to Py. yezoensis mtDNA reported from China (NC_017837; 41,688 bp). However, the Korean Py. yezoensis differed from the Chinese strain in the presence/absence of introns and intronic open reading frames of the ribosomal RNA large subunit gene (rnl) and cytochrome c oxidase subunit 1 gene (cox1). These differences result in large variations between the mtDNA of the two strains of Py. yezoensis. Moreover, the Korean strain had a single copy of the trnfM–trnQ region, as compared to a duplicate copy of Py. yezoensis (NC_017837). Pyropia mtDNA exhibited unique genetic features at the intra- and interspecies levels. Therefore, mtDNA genomics can provide novel knowledge for red algal molecular systematics and help to differentiate between cultivars of Pyropia species with new molecular markers.