Megumu Takahashi

Transient Gene Expression System Established in Porphyra yezoensis Is Widely Applicable in Bangiophycean Algae

The establishment of transient gene expression systems in the marine red macroalga Porphyra yezoensis has been useful for the molecular analysis of cellular processes in this species. However, there has been no successful report about the expression of foreign genes in other red macroalgae, which has impeded the broader understanding of the molecular biology of these species. We therefore examined whether the P. yezoensis transient gene expression system was applicable to other red macroalgae. The results indicated that a codon-optimized GUS, designated PyGUS, and plant-adapted sGFP(S65T) were successfully expressed under the control of the P. yezoensis PyAct1 promoter in gametophytic cells of six Porphyra species and also in Bangia fuscopurpurea, all of which are classified as Bangiophyceae. In contrast, there were no reporter-expressing cells in the Florideophycean algae examined. These results indicate the availability of PyGUS and sGFP as reporters and the 5′ upstream region of the PyAct1 gene as a heterologous promoter for transient gene expression in Bangiophycean algae, which could provide a clue to the efficient expression of foreign genes and transformation in marine red macroalgae.

Visualization of Nuclear Localization of Transcription Factors with Cyan and Green Fluorescent Proteins in the Red Alga Porphyra yezoensis

Transcription factors play a central role in expression of genomic information in all organisms. The objective of our study is to analyze the function of transcription factors in red algae. One way to analyze transcription factors in eukaryotic cells is to study their nuclear localization, as reported for land plants and green algae using fluorescent proteins. There is, however, no report documenting subcellular localization of transcription factors from red algae. In the present study, using the marine red alga Porphyra yezoensis, we confirmed for the first time successful expression of humanized fluorescent proteins (ZsGFP and ZsYFP) from a reef coral Zoanthus sp. and land plant-adapted sGFP(S65T) in gametophytic cells comparable to expression of AmCFP. Following molecular cloning and characterization of transcription factors DP-E2F-like 1 (PyDEL1), transcription elongation factor 1 (PyElf1) and multiprotein bridging factor 1 (PyMBF1), we then demonstrated that ZsGFP and AmCFP can be used to visualize nuclear localization of PyElf1 and PyMBF1. This is the first report to perform visualization of subcellular localization of transcription factors as genome-encoded proteins in red algae.

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 Cδ1 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.

Transient Transformation of Red Algal Cells: Breakthrough Toward Genetic Transformation of Marine Crop Porphyra Species

Genetic transformation is a powerful tool not only for elucidating the functions and regulatory mechanisms of genes involved in various physiological events but also for establishing organisms that efficiently produce biofuels and medically functional materials or carry stress tolerance in face of uncertain environmental conditions (Griesbeck et al., 2006; Torney et al., 2007; Bhatnagar-Mathur et al., 2008). Eukaryotic algae classified into microalgae and macroalgae (seaweeds) are highly diverse photosynthetic plants that are utilized as human food and animal feed as well as sources of valuable compounds such as fatty acids, pigments, vitamins and polysaccharides (Hallmann, 2007; Sugawara et al, 2011). Because of their importance in ecology and industry, algae are now considered promising organisms for economical and industrial applications and are thus a target of genetic transformation (Walker et al., 2005; Hallmann, 2007; Blouin et al., 2011). To date, genetic transformation has succeeded in microalgae; thus, stably transformed microalgae are now employed to produce recombinant antibodies, vaccines, or bio-hydrogen as well as to analyze the gene functions targeted for engineering (Sun et al., 2003; Zorin et al., 2009; Specht et al., 2010; Wu et al., 2010). However, it has proven difficult to establish transgenic macroalgae, which has hampered understanding their gene functions in various physiological regulations and also their utilization in biotechnological applications. The red macroalga Porphyra yezoensis is the most popular sea crop in Japan with economical and pharmaceutical importance as the source of foods such as “nori” and pharmacological regents such as the sulfated polysaccharide “porphyran”, which has anti-tumor and antiallergic activities (Noda et al., 1990; Zemke-White & Ohno, 1999; Ishihara et al., 2005). Recently, non-beneficial climate change due to global warming has decreased the quality and yield of P. yezoensis at algal farms by enhancing discoloration and red rot disease caused by fungal infection (Kakinuma et al., 2008; Park et al., 2000). Although breeding of P. yezoensis by traditional selection and crossing methods has progressed to obtain strains showing high growth rates and economically valuable characteristics, these methods have limitations in terms of the isolation of strains carrying heat-stress tolerance or disease

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.

Photosynthesis-dependent Ca2+ influx and functional diversity between phospholipases in the formation of cell polarity in migrating cells of red algae

Unicellular spore cells, designated as monospores (also called archeospores), are well known as migrating plant cells, in which establishment of the anterior-posterior axis directs asymmetrical distribution of F-actin. Since the mechanisms of cell polarity formation are not yet fully elucidated in monospores, we investigated the roles of phosphoinositide signaling systems and Ca2+ mobilization in migration. Although we have already found the critical involvement of phosphatidylinositol 3-kinase in the establishment of cell polarity, we recently demonstrated the important roles of extracellular Ca2+ influx, phospholipase C (PLC) and phospholipase D (PLD). The remarkable characteristics of these factors are that Ca2+ influx depends on photosynthetic activity and that PLC and PLD play roles in the establishment and maintenance of cell polarity, respectively. These findings could provide new insight into the regulation of migration in eukaryotic cells.