Red Seaweed Pyropia Yezoensis Research Articles

Characterization of histone acetyltransferases and deacetylases and their roles in response to dehydration stress in Pyropia yezoensis (Rhodophyta).

Histone acetylation is one of the most pivotal epigenetic mechanisms in eukaryotes and has been tightly linked to the regulation of various genes controlling growth, development and response to environmental stresses in both animals and plants. Till date, the association of histone acetylation to dehydration stress in red algae and genes encoding the enzymes responsible for histone acetylation: histone acetyltransferases (HATs) or histone deacetylases (HDACs), remains largely unknown. In this study, in silico analysis of the red seaweed Pyropia yezoensis identified 6 HAT genes and 10 HDAC genes. These genes displayed good synteny in genome loci with their Pyropia haitanensis orthologs except for a putative gene duplication event in HDAC and a loss of one HAT gene in P. yezoensis. According to the conserved domains and phylogenetic analysis, they encoded three GCNA5-, one TAFII250- and one MYST-HAT, as well as five HDA1-and five SIRT-HDACs. The sirtuin-domain of Py06502 harbored a ~100 aa insert and interestingly, this insertion was specifically observed in Bangiales species. Two nuclear-localized HATs were transcriptionally up-regulated at the early stage of dehydration and so were two nuclear HDA1s when moderate dehydration started, suggesting their potential roles in modulating downstream gene expression to facilitate dehydration adaptation by changing histone acetylation patterns on relevant regulatory elements. This was experimentally confirmed by the increased decline in photosynthesis efficiency during dehydration when HAT and HDAC activities were inhibited by SAHA and MB-3, respectively. Transcriptional patterns of multiple dehydration-responsive genes after water loss were strongly affected by MB-3 or SAHA treatment. This study provides the first insight into the regulation and function of HAT/HDAC during stress adaptation in red algae.

Low temperature causes discoloration by repressing growth and nitrogen transporter gene expression in the edible red alga Pyropia yezoensis

The reduced availability of nitrogen sources in seawater leads to discoloration of the edible red seaweed Pyropia yezoensis and induces the expression of genes encoding ammonium, nitrate and urea transporters. In the present study, we demonstrate that low temperatures can also cause discoloration of this economically important seaweed. Thus, we addressed regulatory mechanisms of cold-inducible discoloration. When P. yezoensis thalli were incubated at 0, 5 and 10°C, the thalli exhibited retarded growth and discoloration, along with reduced phycoerythrin contents. Fertilization with nitrogen sources did not recover this discoloration at 0°C, suggesting that defects in nitrogen absorption cause low temperature-induced discoloration. The expression levels of many nitrogen transporter genes were reduced at low temperature. We propose that low temperature-mediated repression of ammonium, nitrate and urea transporter gene expression promotes the reduced absorption of nitrogen sources in P. yezoensis, thereby leading to discoloration. This process is different from the well-known mechanism underlying discoloration in P. yezoensis under nitrogen-deficient conditions at normal culture temperature.

Thallus sectioning as an efficient monospore release method in Pyropia yezoensis (Bangiales, Rhodophyta)

The red seaweed Pyropia yezoensis is a marine crop of important economic value and is widely cultivated in the coastal areas of northern China. The current seedling-raising system relies on the germination of conchospores, a process not fast enough to meet the increasing demand from farmers. In this study, we developed a monospore-dependent seedling method based on the asexual reproduction of the Pyropia thallus. The Pyropia thallus was physically sectioned into small pieces (microthalli) that were cultivated at 15 °C. The algal cells in the microthalli became morphologically condensed, underwent cell division, and then developed into monospores on the 5th day. The monospores were able to attach to the seeding rope in 24 h and germinated into healthy thalli. To optimize the efficiency of monospore release, we tested the effect of temperature and the size and original positions of the microthalli as well as the age of the mother thallus. Microthalli with a size of 30–50 cells from the middle and apex of 21-day-old thalli cultivated at 15 °C yielded the optimum production of monospores. Theoretically, in this thallus-to-thallus seeding strategy, 0.1 g of thallus could produce at least 107 monospores, with the same number of offspring thallus seedlings. Taking all of these advantages together, including the high efficiency, short time, low cost and easy operation, this physical sectioning method could serve as a promising seed source especially for new cultivars with superior traits in Pyropia farming and rapid replenishment of seedlings when thalli undergo disastrous diseases.

A unique life cycle transition in the red seaweed Pyropia yezoensis depends on apospory

Plant life cycles consist of two temporally separated stages: a haploid gametophyte and a diploid sporophyte. In plants employing a haploid–diploid sexual life cycle, the transition from sporophyte to gametophyte generally depends on meiosis. However, previous work has shown that in the red seaweed Pyropia yezoensis, this transition is independent of meiosis, though how and when it occurs is unknown. Here, we explored this question using transcriptomic profiling of P. yezoensis gametophytes, sporophytes, and conchosporangia parasitically produced on sporophytes. We identify a knotted-like homeobox gene that is predominately expressed in the conchosporangium and may determine its identity. We also find that spore-like single cells isolated from the conchosporangium develop directly into gametophytes, indicating that the gametophyte identity is established before the release of conchospores and prior to the onset of meiosis. Based on our findings, we propose a triphasic life cycle for P. yezoensis involving production of gametophytes by apospory.

Functional Characterization and Evolutionary Analysis of Glycine-Betaine Biosynthesis Pathway in Red Seaweed Pyropia yezoensis.

The red seaweed Pyropia yezoensis is an ideal research model for dissecting the molecular mechanisms underlying its robust acclimation to abiotic stresses in intertidal zones. Glycine betaine (GB) was an important osmolyte in maintaining osmotic balance and stabilizing the quaternary structure of complex proteins under abiotic stresses (drought, salinity, etc.) in plants, animals, and bacteria. However, the existence and possible functions of GB in Pyropia remain elusive. In this study, we observed the rapid accumulation of GB in desiccated Pyropia blades, identifying its essential roles in protecting Pyropia cells against severe osmotic stress. Based on the available genomic and transcriptomic information of Pyropia, we computationally identified genes encoding the three key enzymes in the GB biosynthesis pathway: phosphoethanolamine N-methyltransferase (PEAMT), choline dehydrogenase (CDH), and betaine aldehyde dehydrogenase (BADH). Pyropia had an extraordinarily expanded gene copy number of CDH (up to seven) compared to other red algae. Phylogeny analysis revealed that in addition to the one conservative CDH in red algae, the other six might have originated from early gene duplication events. In dehydration stress, multiple CDH paralogs and PEAMT genes were coordinating up-regulated and shunted metabolic flux into GB biosynthesis. An elaborate molecular mechanism might be involved in the transcriptional regulation of these genes.

Difference in Nitrogen Starvation-Inducible Expression Patterns among Phylogenetically Diverse Ammonium Transporter Genes in the Red Seaweed <i>Pyropia yezoensis</i>

Nitrogen deficiency induces senescence and the expression of genes encoding ammonium transporters (AMTs) in terrestrial plants where the AMT family is subdivided into AMT1 and AMT2 subfamilies. Nitrogen starvation in the red seaweed Pyropia yezoensis causes senescence-like discoloration. In this study, we identified five genes in P. yezoensis encoding AMT domain-containing proteins, which were phylogenetically categorized into the AMT1 subfamily. We also found a gene encoding a Rhesus protein (Rh) that was related to, but diverged from, AMTs. Moreover, our phylogenetic analysis showed that AMT domain-containing proteins from micro- and macro-algae belonged to either the AMT1 or Rh subfamily, indicating the absence of AMT2 in algae. Gene expression analyses revealed the presence of gametophyte- and sporophyte-specific AMT1 genes that were up-regulated transiently and continually, respectively, under nitrogen-deficient conditions. In addition, up-regulated sporophyte-specific gene expression was suppressed when nitrogen was resupplied. Accordingly, an expansion of the ancient AMT gene has produced AMT1 functional variants differing in temporal and nitrogen starvation-inducible expression patterns during the life cycle of P. yezoensis. These findings help elucidate the unique nutrition starvation responses involving functionally diverse AMT1 and Rh subfamilies in red seaweed.

A Simple Procedure to Observe Phototropic Responses in the Red Seaweed Pyropia yezoensis.

The marine red seaweed Pyropia yezoensis exhibits phototropic responses in gametophyte and conchosporangia phases, but not in sporophytes. These responses are easily monitored with a simple culturing box that has one side open to allow for unilateral light irradiation within an incubator. Confirmation of phototropic responses is achieved by changing the direction of unilateral light irradiation via rotation of the culture dishes clockwise 90°.

Carotenoid Profiling of a Red Seaweed Pyropia yezoensis: Insights into Biosynthetic Pathways in the Order Bangiales.

Carotenoids are natural pigments that contribute to light harvesting and photo-protection in photosynthetic organisms. In this study, we analyzed the carotenoid profiles, including mono-hydroxy and epoxy-carotenoids, in the economically valuable red seaweed Pyropia yezoensis, to clarify the detailed biosynthetic and metabolic pathways in the order Bangiales. P. yezoensis contained lutein, zeaxanthin, α-carotene, and β-carotene, as major carotenoids in both the thallus and conchocelis stages. Monohydroxy intermediate carotenoids for the synthesis of lutein with an ε-ring from α-carotene, α-cryptoxanthin (β,ε-caroten-3’-ol), and zeinoxanthin (β,ε-caroten-3-ol) were identified. In addition, β-cryptoxanthin, an intermediate in zeaxanthin synthesis from β-carotene, was also detected. We also identified lutein-5,6-epoxide and antheraxanthin, which are metabolic products of epoxy conversion from lutein and zeaxanthin, respectively, by LC-MS and 1H-NMR. This is the first report of monohydroxy-carotenoids with an ε-ring and 5,6-epoxy-carotenoids in Bangiales. These results provide new insights into the biosynthetic and metabolic pathways of carotenoids in red seaweeds.

Discolored Red Seaweed Pyropia yezoensis with Low Commercial Value Is a Novel Resource for Production of Agar Polysaccharides.

The red seaweed Pyropia yezoensis has been demonstrated to be a novel resource for the production of high-quality agar. P. yezoensis is grown for the food industry in large-scale Japanese mariculture operations. However, discolored P. yezoensis is mostly discarded as an industrial waste, although it has some kind of utility values. Here, we evaluated the utility of discolored P. yezoensis as a resource for agar production. The quality of agar from the discolored seaweed was comparable to that from normal seaweed. In addition, as a distinguishing characteristic, agar yield was higher from discolored seaweeds than from normal types. Moreover, we successfully used agar from discolored P. yezoensis for bacterial plate media and DNA electrophoresis gels without agarose purification. Thus, our results demonstrate that discolored P. yezoensis is suitable for agar production and use in life science research. Diverting discolored P. yezoensis from disposal to agar production provides a solution to the current industrial waste problem in mariculture, as well as a secure source of agar for research purposes.

Optimization of yield and quality of agar polysaccharide isolated from the marine red macroalga Pyropia yezoensis

The marine red seaweed Pyropia yezoensis is grown on a large scale in Japan using mariculture for production of nori sheets. Here, we optimized the isolation of agar from P. yezoensis and evaluated its yield, physical properties, and product applications. An alkali pre-treatment of seaweeds before agar extraction increased the weight-average molecular weight of the agar and promoted the conversion of L-galactose sulfate to 3,6-anhydrogalactose, which in turn reduced the sulfate contents, thus altering the gel strength of the agar. This ability to adjust agar quality by altering the alkali concentration during material pre-treatment allowed production of agar with properties similar to those of agar from the red seaweed Gelidium sp., which is widely used for industrial agar applications. We demonstrate the suitability of P. yezoensis agar in production of solid plates for bacterial growth. In addition, the P. yezoensis agar was particularly useful as a gel material, with the capacity for excellent size separation of DNA by electrophoresis even without agarose purification, because of quite low contents of sulfate. These findings support the applicability and usefulness of P. yezoensis for agar production. The established large-scale cultivation methods for P. yezoensis can generate sufficient biomass to produce agar to support medical and biological studies.

Oxidative Stress Promotes Asexual Reproduction and Apogamy in the Red Seaweed Pyropia yezoensis.

The marine red seaweed Pyropia yezoensis has a haploid-diploid life cycle wherein two heteromorphic generations, a haploid gametophyte and a diploid sporophyte, are reciprocally generated from conchospores and carpospores, respectively. When we treated gametophytic blades of P. yezoensis with H2O2, discharge of asexual monospores was accelerated, resulting in increased numbers of gametophytic clones. Production of sporophytes without fertilization of male and female gametes was also observed. These findings indicate that oxidative stress can induce vegetative cells to develop into monospores that produce gametophytes asexually and can sometimes prompt carpospores to develop into sporophytes. The discovery of oxidative stress-triggered asexual reproduction and -apogamy will stimulate progress in studies of life-cycle regulation in P. yezoensis.

A technical breakthrough close at hand: feasible approaches toward establishing a gene-targeting genetic transformation system in seaweeds

The lack of a genetic transformation system that allows integration of a foreign gene into the genome is a serious hindrance to progress in biological research on seaweeds. In contrast, physiological research in seaweeds has a long history of focus on the establishment of cell polarity and multicellularity (Brownlee and Bouget, 1998; Hable and Hart, 2010). Today, nuclear genomes have been recently sequenced for many species of seaweeds and there is now a significant accumulation of genomic information in the form of expressed sequence tag data in public and private databases for analyses (see Mikami, 2014). Although physiological and molecular biological approaches have the potential to allow genome-wide and comprehensive analyses of factors involved in seaweed development, the lack of a genetic transformation system hinders functional identification of genes encoding regulatory components of seaweed development. This is impeding the application of the molecular biological knowledge in industrial and economic activities related to seaweed. Because genetic manipulation systems are indispensable for the functional analysis of genes, it is necessary to develop reverse genetic methods for gene knock-out, knock-in, or replacement in a variety of seaweeds via either homologous recombination or random integration of foreign DNA with a marker to identify its integration site in the genome. To accelerate progress toward establishing genetic transformation methods, there are four important issues to be resolved: (1) creating expression constructs particularly suited to seaweed cells; (2) transferring the expression constructs into cells; (3) ensuring integration of expression constructs into the genome; and (4) selecting genetically transformed cells. It has already been confirmed that combining codon-optimized coding regions and endogenous strong promoters results in high-efficiency expression in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). Seaweed genomes generally have high GC content and thus coding sequences should be biased toward higher amounts of G and C. Moreover, the Cauliflower mosaic virus (CaMV) 35S RNA promoter that has been used for foreign gene expression in seaweeds prior to establishment of the transient gene expression has rather low activity in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). But importantly, neither the codon optimization nor endogenous promoters yet tested could enhance the expression of foreign genes in seaweeds (Mikami et al., 2011). These findings clearly indicate that codon-optimized coding regions directed by strong endogenous promoters will be required elements of the design of constructs that can be expressed efficiently in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). This strategy has already been applied to a wide variety of red seaweeds (Hirata et al., 2011a,b; Son et al., 2011). Moreover, the second requirement for genetic transformation, to transfer expression constructs into cells, has been resolved using particle bombardment technology in red seaweeds (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013) and microinjection in Ectocarpus siliculosus (Farnham et al., 2013). However, neither electroporation nor glass bead methods have yet been successful for transferring DNA constructs into seaweed cells. Our preliminary data indicated that the glass bead method and various gene transfer regents designed for mammalian cells were ineffective for cell wall–less protoplasts and monospores from the red seaweed Pyropia yezoensis. Taken together, the third and fourth issues—to ensure integration of expression constructs into the genome, and select genetically transformed cells, respectively—still have to be resolved to establish nuclear genetic transformation.