Development of koji by culturing Aspergillus oryzae on nori (Pyropia yezoensis).

Koji is a traditional fermentation culture medium, based on Aspergillus oryzae, which is commonly used in the manufacture process of Japanese fermented products such as soy sauce, miso, and sake, and promote enzymatic degradation. Koji is usually prepared by culturing a mold on cereals such as wheat flour, soybean, or rice, but that cultured on seaweeds has not been developed yet. This study prepared the koji by culturing A. oryzae on seaweed nori (dried piece of Pyropia yezoensis), and, then, characterized on this nori koji. The nori koji contained 0.85 µg N-acetylglucosamine, estimated as 6.1 µg mold cells, per gram dry matter and showed various kind of enzymatic activities in glycosidase, protease, and phosphatase as well as traditional soy sauce koji and rice koji. The suitability of these characteristics for degradation of nori was tested on nori sauce culture with and without the addition of the nori koji. After 167 days of culture, the fermentation tank with the nori koji showed over 74% recovery of supernatant while that without the nori koji had less than 57% recovery. The supernatant of culture mashes contained more than two times larger quantity of total nitrogen compounds in nori koji test group against control group. The present study prepared koji on seaweed nori for the first time and demonstrated its advantages to shorten the culture period and increase taste value in nori sauce manufacture. Development of seaweed koji enables a method to prepare cereal allergen free fermented sauces from seaweeds.

Polyploid lineages in the genus Porphyra.

Whole genome duplication is now accepted as an important evolutionary force, but the genetic factors and the life history implications affecting the existence and abundance of polyploid lineages within species are still poorly known. Polyploidy has been mainly studied in plant model species in which the sporophyte is the dominant phase in their life history. In this study, we address such questions in a novel system (Porphyra, red algae) where the gametophyte is the dominant phase in the life history. Three Porphyra species (P. dioica, P. umbilicalis, and P. linearis) were used in comparisons of ploidy levels, genome sizes and genetic differentiation using flow cytometry and 11 microsatellite markers among putative polyploid lineages. Multiple ploidy levels and genome sizes were found in Porphyra species, representing different cell lines and comprising several cytotype combinations among the same and different individuals. In P. linearis, genetic differentiation was found among three polyploid lineages: triploid, tetraploid and mixoploids, representing different evolutionary units. We conclude that the gametophytic phase (n) in Porphyra species is not haploid, contradicting previous theories. New hypotheses for the life histories of Porphyra species are discussed.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Morphological and photosynthetic variations in the process of spermatia formation from vegetative cells in Porphyra yezoensis 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.

Ca2+ influx and phosphoinositide signalling are essential for the establishment and maintenance of cell polarity in monospores from the red alga Porphyra yezoensis.

The asymmetrical distribution of F-actin directed by cell polarity has been observed during the migration of monospores from the red alga Porphyra yezoensis. The significance of Ca2+ influx and phosphoinositide signalling during the formation of cell polarity in migrating monospores was analysed pharmacologically. The results indicate that the inhibition of the establishment of cell polarity, as judged by the ability of F-actin to localize asymmetrically, cell wall synthesis, and development into germlings, occurred when monospores were treated with inhibitors of the Ca2+ permeable channel, phospholipase C (PLC), diacylglycerol kinase, and inositol-1,4,5-trisphosphate receptor. Moreover, it was also found that light triggered the establishment of cell polarity via photosynthetic activity but not its direction, indicating that the Ca2+ influx and PLC activation required for the establishment of cell polarity are light dependent. By contrast, inhibition of phospholipase D (PLD) prevented the migration of monospores but not the asymmetrical localization of F-actin. Taken together, these findings suggest that there is functional diversity between the PLC and PLD signalling systems in terms of the formation of cell polarity; the former being critical for the light-dependent establishment of cell polarity and the latter playing a role in the maintenance of established cell polarity.

Ammonium induced expression of the red algal chloroplast gene Ycf18, a putative homolog of the cyanobacterial NblA gene involved in nitrogen deficiency-induced phycobilisome degradation.

In cyanobacteria, nutrient deficiency-induced phycobilisome degradation is controlled by the NblA gene. Red algae also have an NblA-related gene, Ycf18, in their chloroplast genomes. To elucidate the role of Ycf18, the expression pattern of Ycf18 in a red alga, Porphyra yezoensis, was investigated. Ycf18 expression was low in nitrate medium, but was greatly promoted in ammonium medium. Nitrogen starvation caused bleaching, but did not affect the expression of Ycf18. The responses of Ycf18 to nitrogen-starvation and the supply of ammonium were distinct from those of NblA, suggesting that Ycf18 has a role other than the regulation of phycobilisome degradation.

Visualization of phosphoinositides via the development of the transient expression system of a cyan fluorescent protein in the red alga Porphyra yezoensis.

Phosphoinositides (PIs) play important roles in signal transduction pathways and the regulation of cytoskeleton and membrane functions in eukaryotes. Subcellular localization of individual PI derivative is successfully visualized in yeast, animal, and green plant cells using PI derivative-specific pleckstrin homology (PH) domains fused with a variety of fluorescent proteins; however, expression of fluorescent proteins has not yet been reported in any red algal cells. In the present study, we developed the system to visualize these PIs using human PH domains fused with a humanized cyan fluorescent protein (AmCFP) in the red alga Porphyra yezoensis. Plasma membrane localization of AmCFP fused with the PH domain from phospholipase Cdelta1 and Akt1, but not Bruton's tyrosine kinase, was observed in cell wall-free monospores, demonstrating the presence of phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4-bisphosphate in P. yezoensis cells. This is the first report of the successful expression of fluorescent protein and the monitoring of PI derivatives in red algal cells. Our system, based on transient expression of AmCFP, could be applicable for the analysis of subcellular localization of other proteins in P. yezoensis and other red algal cells.

Establishment and cultivation of cell line HB with high temperature resistance and fast growth in Porphyra yezoensis.

A new breeding technology with cell culture in Porphyra yezoensis was studied, to establish a system of fast breeding with cell engineering in Porphyra. By means of somatic cell isolation and multiple clone technique, 4 pure cell-lines (HA, HB, HC, HD) have been established in Porphyra yezoensis. With purely culturing the cell seedlings and conchocelis filaments of the cell lines, their growth rate and resistance to higher temperature were measured. Among cell line HA, HB, HC, HD, HB was the best for resisting higher temperature (at 19 degrees C, 21 degrees C, 23 degrees C, 25 degrees C). Also the growth rate of HB was faster than others. In 1998-2000, the HB was cultivated at sea field of Haifeng in Qidong County, Jiangsu Province. The yield of HB was higher than that of local cultivar. So the HB might be a good cell line for both resisting higher temperature and faster growth. It showed the breeding with cell culture was a fast breeding method.