Toshiki Uji

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.

Ethylene regulation of sexual reproduction in the marine red alga Pyropia yezoensis (Rhodophyta)

Plant growth regulators (PGRs) play a pivotal role in vascular plants, regulating growth, development, and stress responses; however, the role of PGRs in algae remains largely unexplored. Here, the role of ethylene, a simple plant growth regulator, was demonstrated in sexual reproduction of the marine red alga Pyropia yezoensis. Application of the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) promoted the formation of spermatia and zygotospores in the gametophytes as well as ethylene production, whereas the growth rate was repressed in comparison to gametophytes not treated with ACC. In addition, gametophytes treated with ACC and mature gametophytes showed enhanced tolerance to oxidative stress. Gene expression profiles revealed upregulation of genes involved in cell division and stress response in gametophytes treated with ACC and in mature gametophytes. These results indicate that ethylene plays an important role in the regulation of gamete formation and protection against stress-induced damage during the sexual reproductive stage. Considered together, these findings demonstrate that ethylene is involved in regulating the switching from a vegetative to a sexual reproductive phase in P. yezoensis.

Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta).

The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.

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

A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific.

A Codon-Optimized Bacterial Antibiotic Gene Used as Selection Marker for Stable Nuclear Transformation in the Marine Red Alga Pyropia yezoensis

Marine macroalgae play an important role in marine coastal ecosystems and are widely used as sea vegetation foodstuffs and for industrial purposes. Therefore, there have been increased demands for useful species and varieties of these macroalgae. However, genetic transformation in macroalgae has not yet been established. We have developed a dominant selection marker for stable nuclear transformation in the red macroalga Pyropia yezoensis. We engineered the coding region of the aminoglycoside phosphotransferase gene aph7″ from Streptomyces hygroscopicus to adapt codon usage of the nuclear genes of P. yezoensis. We designated this codon-optimized aph7″ gene as PyAph7. After bombarding P. yezoensis cells with plasmids containing PyAph7 under the control of their endogenous promoter, 1.9 thalli (or individuals) of hygromycin-resistant strains were isolated from a 10-mm square piece of the bombarded thallus. These transformants were stably maintained throughout the asexual life cycle. Stable expression of PyAph7was verified using Southern blot analysis and genomic PCR and RT-PCR analyses. PyAph7 proved to be a new versatile tool for stable nuclear transformation in P. yezoensis.

Stable Nuclear Transformation System for the Coccolithophorid Alga Pleurochrysis carterae

Of the three dominant marine microalgal groups, dinoflagellates and diatoms can undergo genetic transformation; however, no transformation method has been established for haptophytes to date. Here, we report the first stable genetic transformation of a coccolithophore, Pleurochrysis carterae, by means of polyethylene glycol (PEG)-mediated transfer of a bacterial hygromycin B-resistance gene. Together with the novel transient green fluorescent protein (GFP) expression system, this approach should facilitate further molecular-based research in this phylum.

Characterization of the Sporophyte-Preferential Gene Promoter from the Red Alga Porphyra yezoensis Using Transient Gene Expression

The life cycle of plants entails an alternation of generations, the diploid sporophyte and haploid gametophyte stages. There is little information about the characteristics of gene expression during each phase of marine macroalgae. Promoter analysis is a useful method for understanding transcriptional regulation; however, there is no report of promoter analyses in marine macroalgae. In this study, with the aim of elucidating the differences in the transcriptional regulatory mechanisms between the gametophyte and sporophyte stages in the marine red alga Porphyra yezoensis, we isolated the promoter from the sporophyte preferentially expressed gene PyKPA1, which encodes a sodium pump, and analyzed its promoter using a transient gene expression system with a synthetic β-glucuronidase (PyGUS) reporter. The deletion of −1432 to −768 relative to the transcription start site resulted in decreased GUS activity in sporophytes. In contrast, deletion from −767 to −527 increased GUS activity in gametophytes. Gain-of-function analyses showed that the −1432 to −760 region enhanced the GUS activity of a heterologous promoter in sporophytes, whereas the −767 to −510 region repressed it in gametophytes. Further mutation and gain-of-function analyses of the −767 to −510 region revealed that a 20-bp GC-rich sequence (−633 to −614) is responsible for the gametophyte-specific repressed expression. These results showed that the sporophyte-specific positive regulatory region and gametophyte-specific negative regulatory sequence play a crucial role in the preferential expression of PyKPA1 in P. yezoensis sporophytes.

Changes in membrane fluidity and phospholipase D activity are required for heat activation of PyMBF1 in Pyropia yezoensis (Rhodophyta)

Multiprotein bridging factor 1 (MBF1) is a highly conserved transcriptional co-activator involved in the regulation of diverse processes, such as environmental stress responses. We recently identified a novel MBF1 gene, PyMBF1, from the marine red alga Pyropia yezoensis. In this study, quantitative real-time PCR analysis revealed that PyMBF1 transcripts were upregulated in P. yezoensis cells during exposure to oxidative and heat stresses. We also examined heat signaling in P. yezoensis cells by monitoring the accumulation of PyMBF1 transcripts. Heat activation of PyMBF1 was inhibited by the membrane rigidifier dimethylsulfoxide, whereas it was induced without heat stress by the membrane fluidizer benzyl alcohol (BA). Induction of PyMBF1 transcripts by heat and BA was inhibited by 1-butanol, an inhibitor of phospholipase D (PLD). The results suggest that the heat activation of PyMBF1 requires membrane fluidization and activation of PLD. These findings provide an initial step toward understanding heat signaling in marine red algae.