Marine Red Alga Porphyra Yezoensis Research Articles

Functional expression of an animal type-Na+-ATPase gene from a marine red seaweed Porphyra yezoensis increases salinity tolerance in rice plants

Despite lacking Na+-ATPase as a sodium pump in vascular plants, a gene encoding KPA (K+ P-type ATPase), a putative animal type-Na+/K+-ATPase, has been isolated from the marine red alga Porphyra yezoensis and designated PyKPA1. To characterize the properties of PyKPA1 and also to confirm its ability to confer salinity tolerance in land plants, transgenic rice plants were produced that expressed the full-length PyKPA1 cDNA under the control of cauliflower mosaic virus 35S RNA promoter. We observed transcriptional activation of the transgene, plasma membrane-localization of the gene product fused with green fluorescent protein in onion epidermal cells, and Na+-ATPase activity in the plasma membrane fraction from transgenic rice plants, indicating that PyKPA1 was functionally expressed in rice plants. Transgenic lines were examined in terms of growth in salinity stress conditions, resulting in protection from a decrease in biomass, although growth of control rice plants was repressed. These results demonstrate the utility of a red algal animal type-sodium pump for conferring salinity tolerance to land plants.

Polaribacter porphyrae sp. nov., isolated from the red alga Porphyra yezoensis, and emended descriptions of the genus Polaribacter and two Polaribacter species

Three Gram-negative, non-motile, strictly aerobic strains, designated LNM-20(T), LCM-1 and LAM-13, were isolated from thalli of the marine red alga Porphyra yezoensis. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates were associated with the genus Polaribacter in the family Flavobacteriaceae and were most closely related to Polaribacter dokdonensis DSW-5(T) (96.2 % 16S rRNA gene sequence similarity) and Polaribacter gangjinensis K17-16(T) (95.0 %). The DNA G+C content of the isolates was 28.6-29.2 mol%. DNA-DNA hybridization analysis showed that the isolates belonged to a single species distinct from both of their closest relatives. The only isoprenoid quinone detected was menaquinone-6. The main polar lipids were phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids. The major fatty acids were iso-C15 : 0, iso-C15 : 1ω10c and iso-C15 : 0 3-OH. The phenotypic features of strain LNM-20(T) differed from those of their closest relatives in several regards (colony colour, growth with 1 % NaCl and on TSA plus 2.5 % NaCl, hydrolysis of Tweens 40 and 80, and oxidization of five carbon compounds). On the basis of phylogenetic, chemotaxonomic and phenotypic analysis, the isolates represent a novel species in the genus Polaribacter, for which the name Polaribacter porphyrae sp. nov. is proposed. The type strain is LNM-20(T) ( = LMG 26671(T) = NBRC 108759(T)). Emended descriptions of the genus Polaribacter and P. dokdonensis and P. gangjinensis are also proposed.

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.

Molecular characterization and expression analysis of two Na+/H+ antiporter genes in the marine red alga Porphyra yezoensis

Na+/H+ antiporters are known to play a crucial role in pH and Na+ homeostasis. In the present study, we characterized the molecular structures and expression patterns of two Na+/H+ antiporters from the marine red alga Porphyra yezoensis (designated PySOS1 and PyNhaD). The full-length cDNAs of PySOS1 and PyNhaD were 5122 and 1804 bp, and contained open reading frames (ORFs) of 4773 and 1275 bp, respectively. The deduced amino acid sequences showed high similarity to SOS1 and NhaD from the higher plant Arabidopsis thaliana. PySOS1 and PyNhaD contained conserved sequences found in the cation–proton antiporter. Quantitative real-time PCR analysis revealed that both antiporter genes were expressed in both the gametophyte and sporophyte of P. yezoensis. In addition, mRNA expression of PySOS1 and PyNhaD was simultaneously upregulated by light irradiation, suggesting that coordinated activity between the two is important in pH and Na+ homeostasis under light conditions. Moreover, the expression levels of both genes were partially reduced by the photosynthetic inhibitors DCMU and DBMIB, suggesting that upregulation is linked to photosynthesis-related metabolism. These findings provide an initial step towards understanding Na+/H+ antiporters in marine red algae.

Molecular characterization and expression analysis of sodium pump genes in the marine red alga Porphyra yezoensis

Sodium pumps (EC 3.6.3.9, Na(+)-ATPase), which mediate excretion of Na(+) from the cell, play a crucial role in Na(+) homeostasis in eukaryotic cells. The objective of this study is to understand the Na(+) efflux system in a marine red alga. We identified a novel sodium pump gene, PyKPA2, from the marine red alga Porphyra yezoensis. The amino acid sequence of PyKPA2 shares 65 % identity with PyKPA1, a previously identified P. yezoensis sodium pump. Similar to PyKPA1, PyKPA2 contains conserved sequences for functions such as phosphorylation, ATP binding, and cation binding. Phylogenetic analysis revealed that the two genes cluster with sodium pumps from algae. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that PyKPA1 is expressed preferentially in sporophytes, whereas PyKPA2 is expressed specifically in gametophytes. RT-PCR and quantitative real-time PCR analysis revealed that PyKPA1 and PyKPA2 transcripts were upregulated and downregulated, respectively, in gametophytes during exposure to alkali stress. In addition, transcription of both genes in gametophytes was also induced by cold stress. These results suggest that PyKPA1 and PyKPA2 play an important role in alkali and cold stress tolerance.

Maritalea porphyrae sp. nov., isolated from a red alga (Porphyra yezoensis), and transfer of Zhangella mobilis to Maritalea mobilis comb. nov.

Three Gram-negative, motile, aerobic bacteria were isolated from cultures of the marine red alga Porphyra yezoensis. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel strains were closely related to Maritalea myrionectae CL-SK30(T) (97.9% 16S rRNA gene sequence similarity) and Zhangella mobilis E6(T) (96.2 %). 16S rRNA gene sequence similarity between Z. mobilis E6(T) and M. myrionectae CL-SK30(T) was 97.9%. The DNA G+C contents of the isolates (49.4-50.0 mol%) were similar to those of M. myrionectae DSM 19524(T) (52.3 mol%) and Z. mobilis JCM 15144(T) (50.3 mol%). From these results, it was difficult to differentiate the genus Zhangella from the genus Maritalea. DNA-DNA hybridization demonstrated that the isolates belonged to a single species. The isolates could also be distinguished from M. myrionectae and Z. mobilis on the basis of chemotaxonomic and phenotypic features, including fatty acid composition (particularly C(16:1)ω7c), growth with 6-9% (w/v) NaCl, carbon utilization, oxidation patterns and so on. A novel species of the genus Maritalea is proposed to accommodate the three isolates, with the name Maritalea porphyrae sp. nov. The type strain is LCM-3(T) (=LMG 25872(T)=NBRC 107169(T)). Furthermore, it is proposed that Zhangella mobilis should be transferred from the genus Zhangella to the genus Maritalea, with the name Maritalea mobilis comb. nov. (type strain E6(T)=CGMCC 1.7002(T)=JCM 15144(T)).

Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis

Despite the recent progress of transient gene expression systems in a red alga Porphyra yezoensis by particle bombardment, a stable transformation system has yet to establish in any marine red macrophytes. One of the reasons of the difficulty in genetic transformation in red algae is the lack of systems to select and isolate transformed cells from gametophytic blades. Thus, toward the establishment of the stable transformation system in P. yezoensis , we have developed a procedure by which transiently transformed gametophytic cells were prepared from particle bombarded-gametophytic blade as regeneratable protoplasts. Using mixture of marine bacterial enzymes, yield of protoplasts was high as reported elsewhere; however, these protoplasts did not develop. In contrast, protoplasts prepared from gametophytes treated with allantoin were normally developed, in which the overexpression of a β-glucuronidase reporter gene had no effect on the regeneration of protoplasts. Therefore, the use of allantoin in protoplast preparation sheds a new light on the realization of an efficient isolation and selection of study transformed cells from gametophytic blades.

10.1007/s11183-008-2004-1

In order to investigate the possible effects of the ecological environment on photosynthetic activity and the major light harvesting complex, the oxygen evolution rates and composition of phycobilisome from marine red alga Porphyra yezoensis Ueda and freshwater red alga Compsopogon coeruleus (Balbis) Montagne, which could grow and reproduce under salinity up to 35 ppt, were studied. The results showed that the oxygen evolution rate of P. yezoensis in seawater was significantly higher than that of C. coeruleus in freshwater, and P. yezoensis tolerated inorganic ions at a relatively higher concentration than C. coeruleus. Moreover, the phycoerythrin (PE) of P. yezoensis was R-phycoerythrin containing α, β, and γ subunits comprised phycoerythrobilin and phycourobilin. In contrast, the PE from C. coeruleus consisted of α, β, and γ subunits comprised only phycoerythrobilin but not phycourobilin, suggesting that the PE from C. coeruleus was of a new type.

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.

Effects of cell wall synthesis on cell polarity in the red alga Porphyra yezoensis

Polarity is a fundamental cell property essential for differentiation, proliferation and morphogenesis in unicellular and multicellular organisms. We have recently demonstrated that phosphatidylinositol 3-kinase (PI3K) activity is required for the establishment of anterior-posterior axis, leading to asymmetrical localization of F-actin in migrating monospores of the red alga Porphyra yezoensis. We also showed that the formation of the apical-basal axis via adhesion of monospores to the substratum after the cessation of migration requires newly synthesized proteins and does not depend on PI3K activity. However, little is known about the mechanism and regulation of axis conversion during development of monospores. In this addendum, we report our investigation as to the role of the cell wall in axis conversion. Our results indicate that inhibition of cell wall synthesis prevented the development of germlings. Also, defects in the cell wall disrupted the asymmetrical distribution of F-actin and inhibited the adhesion to the substratum that is required for establishment of apical-basal axis. Hence, we conclude that the cell wall is critical for the maintenance of cell polarity in migrating cells, which is indirectly involved in axis conversion via enabling monospores to adhere to the substratum.Addendum to: Li L, Saga N, Mikami K. Phosphatidylinositol 3-kinase activity and asymmetrical accumulation of F-actin are necessary for establishment of cell polarity in the early development of monospores from the marine red alga Porphyra yezoensis. J Exp Bot 2008; 59:3575–86; PMID: 18703492; DOI: 10.1093/jxb/ern207.

Phosphatidylinositol 3-kinase activity and asymmetrical accumulation of F-actin are necessary for establishment of cell polarity in the early development of monospores from the marine red alga Porphyra yezoensis

The polarized distribution of F-actin is important in providing the driving force for directional migration in mammalian leukocytes and Dictyostelium cells, in which compartmentation of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol phosphatase is critical for the establishment of cell polarity. Since monospores from the red alga Porphyra yezoensis are a real example of migrating plant cells, the involvement of the cytoskeleton and PI3K was investigated during their early development. Our results indicate that the asymmetrical localization of F-actin at the leading edge is fixed by the establishment of the anterior–posterior axis in migrating monospores, which is PI3K-dependent and protein synthesis-independent. After migration, monospores adhere to the substratum and then become upright, developing into multicellular thalli via the establishment of the apical–basal axis. In this process, F-actin usually accumulates at the bottom of the basal cell and development after migration requires new protein synthesis. These findings suggest that the establishment of anterior–posterior and apical–basal axes are differentially regulated during the early development of monospores. Our results also indicate that PI3K-dependent F-actin asymmetry is evolutionally conserved in relation to the establishment of cell polarity in migrating eukaryotic cells.

Inheritance pattern of chloroplast and mitochondrial genomes in artificial hybrids ofPorphyra yezoensis(Rhodophyta)

To investigate inheritance of chlorplasts and mitochondria (organelles) in the marine red alga Porphyra yezoensis, cleaved amplified polymorphic sequence (CAPS) profiles of the two strains, TU-2 and KGJ, as parental gametophytes and 44 hybrid sporophytes (i.e. female of TU-2 and male of KGJ) were examined. A carbamyl phosphate synthase small subunit gene (carA) and ribosomal protein S11 gene (rps11)-small subunit ribosomal RNA gene (rns) spacer region were used as molecular markers for the chloroplast and mitochondrial DNAs, respectively. Thirty-eight of 44 (86.4%) conchocelis colonies showed maternal banding patterns, five (11.4%) colonies showed biparental bands and one (2.2%) colony showed paternal bands in CAPS analyses both with chloroplast and mitochondrial markers. These results suggest that organelles of P. yezoensis are inherited uniparentally from the female parent.

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.

Comparison between Compsopogon coeruleus and Porphyra yezoensis in their O2 evolution rates and phycobilisome composition

In order to investigate the possible effects of the ecological environment on photosynthetic activity and the major light harvesting complex, the oxygen evolution rates and composition of phycobilisome from marine red alga Porphyra yezoensis Ueda and freshwater red alga Compsopogon coeruleus (Balbis) Montagne, which could grow and reproduce under salinity up to 35 ppt, were studied. The results showed that the oxygen evolution rate of P. yezoensis in seawater was significantly higher than that of C. coeruleus in freshwater, and P. yezoensis tolerated inorganic ions at a relatively higher concentration than C. coeruleus. Moreover, the phycoerythrin (PE) of P yezoensis was R-phycoerythrin containing alpha, beta, and gamma subunits comprised phycoerythrobilin and phycourobilin. In contrast, the PE from C. coeruleus consisted of alpha, beta, and gamma subunits comprised only phycoerythrobilin but not phycourobilin, suggesting that the PE from C. coeruleus was of a new type.

Factors influencing efficiency of transient gene expression in the red macrophyte Porphyra yezoensis

The marine red alga Porphyra yezoensis has been proposed as a model plant for physiological and genetic studies in seaweeds because of its biological and economical importance. However, the progress of molecular biological studies using gene transfection and genetic transformation systems has been hindered by difficulties in the expression of foreign genes in P. yezoensis cells. To overcome this situation, we developed a transient gene expression system to monitor gene expression in P. yezoensis cells. An artificial β-glucuronidase (GUS) coding region was synthesized to adapt it to the codon usage of P. yezoensis (PyGUS) and then evaluated for efficiency as a reporter of transient gene expression by particle bombardment. We also demonstrated the importance of using the promoter of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from P. yezoensis for efficient expression of PyGUS, because the cauliflower mosaic virus (CaMV) 35S promoter, which has been successfully used for monitoring gene expression in nuclei and chloroplasts of higher plants, was less active in P. yezoensis cells. Therefore, the lack of knowledge about differences in the regulatory machinery of gene expression between P. yezoensis and terrestrial plants seems to be why experimental systems for monitoring gene expression were previously not developed in P. yezoensis. Establishment of the transient gene expression system in P. yezoensis could facilitate biotechnological developments in this organism.

Preparation of recombinant polysaccharide-degrading enzymes from the marine bacterium, Pseudomonas sp. ND137 for the production of protoplasts of Porphyra yezoensis

We previously reported that a marine bacterium, strain ND137, produced the enzymes to make protoplasts from the thalli of a marine red alga Porphyra yezoensis. In this study, the bacterium was classified as Pseudomonas sp. based on the sequence of the gene encoding 16S rRNA and the G + C content of the DNA. The β-1,4-xylanase-, cellulase-, β-1,3-xylanase-, porphyranase-, and β-1,4-mannanase-encoding genes were cloned from the genomic library of Pseudomonas sp. ND137 and expressed in Escherichia coli. The protoplasts from Porphyra yezoensis were produced by treating the thalli with several combinations of these recombinant crude enzymes. Treatment of the Porphyra thalli with any of these enzymes on their own failed to produce protoplasts. However, a mixture of β-1,3-xylanase, porphyranase, and β-1,4-mannanase produced protoplasts, although β-1,4-xylanase and cellulase were not observed to have any effect on protoplast production. These results indicate that among the five recombinant crude enzymes, β-1,3-xylanase, porphyranase, and β-1,4-mannanase are essential for producing protoplasts from the thalli of P. yezoensis. The yield of the protoplasts was 1.4 × 104 cells mg−1 wet weight of the thalli, indicating that these recombinant enzymes can be used efficiently for protoplast production from P. yezoensis.

Characterization of a cDNA encoding a homolog of actin-related protein 4 from a marine red alga Porphyra yezoensis

A cDNA (PyARP4) containing an open reading frame for a protein of 573 amino acids was identified in the marine red alga Porphyra yezoensis. The conceptual PyARP4 protein exhibits significant similarity to actin-related protein (ARP) 4 in the terrestrial plant Arabidopsis. Comparison of the deduced amino acid sequence showed moderate sequence identity (30%) to a conventional actin in P. yezoensis, as seen in comparisons between ARP and conventional actins of other organisms. A putative bipartite nuclear localization signal and an actin motif were found within the PyARP4 amino acid sequence. In a phylogenetic analysis, the PyARP4 was found to cluster with the ARP4 of other organisms. The expression level of PyARP4 did not change significantly among four developmental stages of life cycle and was lower than that of a conventional actin. This cDNA therefore may serve as a useful internal standard in gene expression analyses of differentially expressed genes in P. yezoensis.

Rapid isolation and sequence analysis of the beta-tubulin gene from Porphyra yezoensis (Rhodophyta)

Beta-tubulin, one of the cytoskeletal proteins, has been highly conserved throughout the evolution of eukaryotes. Degenerate PCR and inverse PCR (iPCR) were used to isolate the full-length beta-tubulin gene and its 5′ and 3′-flanking regions (2799bp) from the marine red alga Porphyra yezoensis. This gene, designated as TubB1, is devoid of introns. The canonical cis-acting elements such as TATA box, CAAT box and polyA signal AAUAAA are not found in flanking sequences, but another putative polyA signal CAYTG is found downstream of the stop codon. Comparison of the deduced 458 amino acid sequences shows higher similarity to the Protoflorideophycidae Cyanidioschyzon merolae (82%) than to the red alga Chondrus crispus (79%). Codon bias indicates strong expression of TubB1. Phylogenetic analysis suggests that the beta-tubulin of P. yezoensis and C. merola go together with fungi and not with green plants. These nucleotide sequence data have been deposited in the DDB/EMBL/Genbank databases under the accession number AY221630.

Rapid extraction of high‐quality genomic DNA from Porphyra yezoensis (Bangiales, Rhodophyta)

SUMMARYWe 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.

Isolation of bacteria that decompose major polysaccharides in the cell wall of the marine red alga Porphyra yezoensis and their application for protoplast production

We attempted to screen for bacteria that could decompose major polysaccharides in the cell wall of the marine red alga Porphyra yezoensis from Porphyra-culturing farms to enable simple and high-yield preparation of protoplasts with the crude enzyme from a single bacterial origin. A total of 275 positive bacterial strains were isolated by enrichment culture supplemented with Porphyra powder or xylan. Nine strains were capable of producing protoplasts from Porphyra thalli in a 10-fold concentrated culture broth. These strains were identified as two Flavobacterium spp., one Alteromonas sp., four Acinetobacter spp., and two Vibrio spp. The crude enzymes of these bacteria could release 106 protoplast cells from 0.1 g of Porphyra thalli. The crude enzyme from Alteromonas sp. strain ND137 produced the most protoplasts among the nine strains tested. Moreover, an assay of the crude enzymes from the nine bacterial strains for glycosidase activity against four major polysaccharides (xylan, mannan, porphyran, and cellulose) of P. yezoensis revealed strong decomposing activity against these polysaccharides. Xylanase activity was highest in these glycosidases, suggesting that xylanase might be a very important factor in producing protoplasts from Porphyra thalli.Key words: Porphyra, cell wall, bacteria, decomposing polysaccharide.