Naotsune Saga

BACTERIA THAT INDUCE MORPHOGENESIS IN ULVA PERTUSA (CHLOROPHYTA) GROWN UNDER AXENIC CONDITIONS1

Marine foliaceous green macroalgae such as Ulva lose their typical morphology when cultured aseptically in defined synthetic media. However, after reinfection by certain marine bacteria (isolated from unialgal cultures of Ulva pertusa Kjellman), the organisms regain their typical foliaceous or tubular morphology. To investigate the morphogenesis (MG) induced in U. pertusa by bacteria, we isolated and identified bacteria with MG activity on U. pertusa and studied the distribution of such bacteria in seawater and on various marine macroalgae. We isolated 1555 bacterial strains from 18 species of marine macroalgae (six Chlorophyta, five Phaeophyta, and seven Rhodophyta), from seawater and from sediment collected at the beach at Omaezaki, Shizuoka Prefecture; Japan. Of these, 676 bacterial strains (43.5%) showed MG activity. They were classified into six bacterial groups, Flavobacterium, Vibrio, Pseudomonas, Deleya, Escherichia, and gram‐positive cocci. These bacteria were ubiquitous among the samples and were not specific to U. pertusa. Several plant growth regulators had no MG activity. Filter‐sterilized supernatants of culture media of MG‐active bacteria strains did not induce MG. Cocultivation of Ulva with active bacterial strains is so far the only way to induce the MG effect, which suggests that for MG direct contact between Ulva and the bacterial strain is necessary.

Cryopreservation of clonal gametophytic thalli of Porphyra (Rhodophyta)

Abstract Gametophytic thalli of Porphyra were cryopreserved at liquid nitrogen (LN) temperature with a cryoprotective solution in cryogenic vials. Two clonal cultures of gametophytic thalli of P. yezoensis (TU-1,2) and a clonal gametophytic thalli culture of P. tenera (TU-3) were successfully obtained. These gametophytic thalli cultures were clonally maintained via monospores in the laboratory. When the thalli were pre-frozen below −30°C without cryoprotectants, the survival rate was low. Dimethylsulfoxide (DMSO) considerably decreased cell damage during pre-freezing to −40°C, but did little to decrease the damage upon rapid cooling to the LN temperature. This problem was overcome by using dextran, polyvinylpyrrolidone (PVP) or Ficoll in combination with DMSO. The subsequent survival rate after immersion in LN exceeded 95%. This technique allows for the long-term and facile maintenance of clonal culture of gametophytic thalli of Porphyra.

A METHOD FOR EXTRACTION OF HIGH MOLECULAR WEIGHT DNA FROM THE MACROALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1,2

We developed a method for extraction of DNA from the red alga Porphyra yezoensis Ueda. The method consists of three preparation steps that include CsCl‐gradient ultracentrifugation, cetyl trimethyl ammonium bromide treatment, and a final RNase step. The amount of DNA extracted from 1.5 g of starting material averaged 17.7 μg. The resulting DNA had a high molecular weight, was 25‐166 kb in length, was digested with five common restriction enzymes, and showed no nuclease activity. It was of sufficient quality for construction of genomic libraries.

Isolation of a cDNA encoding a homologue of actin from Porphyra yezoensis (Rhodophyta)

We report the nucleotide sequence of a cDNA encoding an actin from amarine red alga, Porphyra yezoensis Ueda. A cDNA clone wasisolated from a leafy gametophyte cDNA library and analyzed for the sequence.The clone contained an open reading frame for a protein of 373 amino acidswhichexhibits sequence similarity to known actins. The GC content of the thirdposition (83.9%) was much higher than that at the first (56.3%) and second(42.4%) positions. The actin forms a gene family in the P.yezoensis genome. Comparison of the deduced amino acid sequenceshowed higher similarity to the Florideophycidae Chondruscrispus (85%) than to the ProtoflorideophycidaeCyanidioschyzon merolae (70%). The mRNA was detected inboth the leafy gametophytes and filamentous sporophytes. The nucleotidesequence data reported in this paper will appear in theDDBJ/EMBL/GenBank databases under accession number AB039831.

Requisite Morphologic Interaction for Attachment between Ulva pertusa (Chlorophyta) and Symbiotic Bacteria

Abstract In order to understand the morphogenesis-inducing mechanism of Ulva pertusa by symbiotic bacteria, we observed the requisite conditions of bacteria for attachment to U. pertusa for algal morphogenesis. Non-morphogenesis-inducing bacterial mutants derived by ultraviolet irradiation did not attach onto the surface of this alga. Scanning electron microscopic observation during the process of morphogenesis in U. pertusa revealed a network-like structure formed on the algal surface within 1 week after application of bacteria. The bacteria attached onto the alga after 2 weeks of incubation. After this attachment process the morphologic change was observed in U. pertusa.

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.

Porphyra monospore system (Bangiales, Rhodophyta): A model for the developmental biology of marine plants

We have developed an experimental system of cohort monospores from clonal culture of leafy gametophytes in Porphyra yezoensis Ueda (strain TU‐1). This system is quite different from traditional systems for algal protoplast experimentation, which require expensive enzymatic treatment and utilize an ineffective method of preservation. Cohort monospores were obtained by utilizing a mode of asexual reproduction in the culture strain (monospores) and artificial regulation (thallus length, temperature, light, etc.) of monospore release. When the leafy gametophytes that formed monospores were frozen at ‐ 20°C in a cryoprotective solution composed of 5% DMSO and 5% dextran in 100% seawater, about 98% survived for 3 months. When stored at 5°C without cryoprotectants, these leafy gametophytes could be kept without monospore release for 1 week. Maximum monospore yield was about 3000 spores per 100 gametophytes, and germination rate was about 70%, This system will accelerate developmental biology studies in Porphyra.

Cryopreservation of the conchocelis of the marine alga Porphyra yezoensis Ueda (Rhodophyta) in liquid nitrogen

Cryopreservation og the conchocelis of Porphyra yezoensis by the two-step cooling method was attempted. A solution composed of 10% DMSO and 0.5 M sorbitol in 50% seawater had the most favorable cryoprotective effect on the conchocelis. The appropriate cryopreservation procedure was as follows: DMSO was added gradually over a period of 15 min followed by an equilibration period of several minutes. Conchocelis cells were then prefrozen to −40°C at 0.1–1°C/min prior to immersion in LN. After storage in LN, the conchocelis suspension was thawed quickly by agitation of the vial in a water bath at 40°C, and cryoprotectants were washed off by gradual dilution with seawater. The survival was independent of the period of storage at least for 300 days. The survival evaluated by staining with neutral red was approx. 60%. The conchocelis cells stored in LN were able to form colonies, and retained the ability to form conchospores which grew into gametophytic thalli.

Growth and the content of laurinterol and debromolaurinterol in Laurencia okamurae (Ceramiales, Rhodophyta)

The growth of Laurencia okamurae and the content of laurinterol and debromolaurinterol were influenced by various factors. Temperature influenced growth raere te with a maximum at 25°C, regardless of daylength. Maturity depended on temperature rather than daylength; the plants grew without maturing at 15°C, while the plants matured within two weeks at 25°C. The plants were able to grow at salinities of 14–50‰ with maximum growth at 26‰. The salinities growth rash;50‰te reduced with decreasing concentration of nitrate below 1.2 × 10-3 M, and of total phosphate below 7.5× 10-8 M. Bromide concentration had no effect on growth, and the plants grew without bromide. Variation in temperature between 15–25°C and daylength produced no obvious change in laurinterol and debromolaurinterol contents. On the other hand, an increase in salinity led to an increase for both. The plants cultured in a completely artificial medium, modified ASP12NTA, showed a marked drop in their content of these metabolites. An increase in concentrations of nitrate, total phosphate or bromide did not restore the content.

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.