Phospholipase D activation is required for 1-aminocyclopropane 1-carboxylic acid signaling during sexual reproduction in the marine red alga Neopyropia yezoensis (Rhodophyta).

BACKGROUND: 1-aminocyclopropane 1-carboxylic acid (ACC) is the immediate precursor of the plant hormone ethylene. However, recent studies have suggested that ACC also acts as a signaling molecule to regulate development and growth independently from ethylene biosynthesis. In red algae, ACC stimulates the switch from a vegetative to a sexual reproductive phase. However, despite evidence that ACC signaling in plants and algae is widespread, the mechanistic basis of the ACC signaling pathway remains unknown. RESULTS: We demonstrate that exogenous ACC increased the activity of phospholipase D (PLD) and induced the accumulation of PLD transcripts in the marine red alga Neopyropia yezoensis. The product of PLD, the lipid second messenger phosphatidic acid (PA), also increased in response to ACC. Furthermore, the pharmacological inhibition of PLD by 1-butanol blocked ACC-induced spermatangia and carpospore production, but the inactive isomer t-butanol did not. In addition, 1-butanol prevented ACC-induced growth inhibition and inhibited transcript accumulation of genes upregulated by ACC, including extracellular matrix (ECM)-related genes, and alleviated the transcriptional decrease of genes downregulated by ACC, including photosynthesis-related genes. CONCLUSIONS: These results indicate that PLD is a positive regulator of sexual cell differentiation and a negative regulator of growth. This study demonstrates that PLD and its product, PA, are components of ACC signaling during sexual reproduction in N. yezoensis.

Characterization of ACE Inhibitory Peptides Prepared from Pyropia pseudolinearis Protein.

More than 7000 red algae species have been classified. Although most of them are underused, they are a protein-rich marine resource. The hydrolysates of red algal proteins are good candidates for the inhibition of the angiotensin-I-converting enzyme (ACE). The ACE is one of the key factors for cardiovascular disease, and the inhibition of ACE activity is related to the prevention of high blood pressure. To better understand the relationship between the hydrolysates of red algal proteins and the inhibition of ACE activity, we attempted to identify novel ACE inhibitory peptides from Pyropia pseudolinearis. We prepared water soluble proteins (WSP) containing phycoerythrin, phycocyanin, allophycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase. In vitro analysis showed that the thermolysin hydrolysate of the WSP had high ACE inhibitory activity compared to that of WSP. We then identified 42 peptides in the hydrolysate by high-performance liquid chromatography and mass spectrometry. Among 42 peptides, 23 peptides were found in chloroplast proteins. We then synthesized the uncharacterized peptides ARY, YLR, and LRM and measured the ACE inhibitory activity. LRM showed a low IC50 value (0.15 µmol) compared to ARY and YLR (1.3 and 5.8 µmol). In silico analysis revealed that the LRM sequence was conserved in cpcA from Bangiales and Florideophyceae, indicating that the novel ACE inhibitory peptide LRM was highly conserved in red algae.

DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus.

Brown algae are one of the most developmentally complex groups within the eukaryotes. As in many land plants and animals, their main body axis is established early in development, when the initial cell gives rise to two daughter cells that have apical and basal identities, equivalent to shoot and root identities in land plants, respectively. We show here that mutations in the Ectocarpus DISTAG (DIS) gene lead to loss of basal structures during both the gametophyte and the sporophyte generations. Several abnormalities were observed in the germinating initial cell in dis mutants, including increased cell size, disorganization of the Golgi apparatus, disruption of the microtubule network, and aberrant positioning of the nucleus. DIS encodes a TBCCd1 protein, which has a role in internal cell organization in animals, Chlamydomonas reinhardtii, and trypanosomes. Our study highlights the key role of subcellular events within the germinating initial cell in the determination of apical/basal cell identities in a brown alga and emphasizes the remarkable functional conservation of TBCCd1 in regulating internal cell organization across extremely distant eukaryotic groups.

Development of Chromatin Immunoprecipitation for the Analysis of Histone Modifications in Red Macroalga Neopyropia yezoensis (Rhodophyta).

Epigenetic regulation by histone modification can activate or repress transcription through changes in chromatin dynamics and regulates development and the response to environmental signals in both animals and plants. Chromatin immunoprecipitation (ChIP) is an indispensable tool to identify histones with specific post-translational modifications. The lack of a ChIP technique for macroalgae has hindered understanding of the role of histone modification in the expression of genes in this organism. In this study, a ChIP method with several modifications, based on existing protocols for plant cells, has been developed for the red macroalga, Neopyropia yezoensis, that consists of a heterogeneous alternation of macroscopic leaf-like gametophytes and microscopic filamentous sporophytes. ChIP method coupled with qPCR enables the identification of a histone mark in generation-specific genes from N. yezoensis. The results indicate that acetylation of histone H3 at lysine 9 in the 5' flanking and coding regions from generation-specific genes was maintained at relatively high levels, even in generation-repressed gene expression. The use of this ChIP method will contribute significantly to identify the epigenetic regulatory mechanisms through histone modifications that control a variety of biological processes in red macroalgae.

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.

The role of plant hormones on the reproductive success of red and brown algae.

Seaweeds or macroalgae are important primary producers that serve as a habitat for functioning ecosystems. A sustainable production of macroalgae has been maintained by a diverse range of life cycles. Reproduction is the most dynamic change to occur during its life cycle, and it is a key developmental event to ensure the species' survival. There is gradually accumulating evidence that plant hormones, such as abscisic acid and auxin, have a role on the sporogenesis of brown alga (Saccharina japonica). Recent studies reported that 1-aminocylopropane-1-carboxylic acid, an ethylene precursor, regulates sexual reproduction in red alga (Neopyropia yezoensis) independently from ethylene. In addition, these macroalgae have an enhanced tolerance against abiotic and biotic stresses during reproduction to protect their gametes and spores. Herein, we reviewed the current understanding on the regulatory mechanisms of red and brown algae on their transition from vegetative to reproductive phase.

Transcriptome Profiling in the Marine Red Alga Neopyropia yezoensis Under Light/Dark Cycle.

Many organisms are subjected to a daily cycle of light and darkness, which significantly influences metabolic and physiological processes. In the present study, Neopyropia yezoensis, one of the major cultivated seaweeds used in "nori," was harvested in the morning and evening during light/dark treatments to investigate daily changes in gene expression using RNA-sequencing. A high abundance of transcripts in the morning includes the genes associated with carbon-nitrogen assimilations, polyunsaturated fatty acid, and starch synthesis. In contrast, the upregulation of a subset of the genes associated with the pentose phosphate pathway, cell cycle, and DNA replication at evening is necessary for the tight control of light-sensitive processes, such as DNA replication. Additionally, a high abundance of transcripts at dusk encoding asparaginase and glutamate dehydrogenase imply that regulation of asparagine catabolism and tricarboxylic acid cycle possibly contributes to supply nitrogen and carbon, respectively, for growth during the dark. In addition, genes encoding cryptochrome/photolyase family and histone modification proteins were identified as potential key players for regulating diurnal rhythmic genes.

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.

Corrigendum: Sexual Reproduction via a 1-Aminocyclopropane-1-Carboxylic Acid-Dependent Pathway Through Redox Modulation in the Marine Red Alga Pyropia yezoensis (Rhodophyta).

[This corrects the article DOI: 10.3389/fpls.2020.00060.].

Sexual Reproduction via a 1-Aminocyclopropane-1-Carboxylic Acid-Dependent Pathway Through Redox Modulation in the Marine Red Alga Pyropia yezoensis (Rhodophyta).

The transition from the vegetative to sexually reproductive phase is the most dynamic change to occur during a plant's life cycle. In the present study, we showed that the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) induces sexual reproduction in the marine red alga Pyropia yezoensis independently from ethylene. Exogenous application of ACC, which contains a three membered carbocyclic ring, promoted the formation of spermatia and carporspores in gametophytes, whereas ethephon, an ethylene-releasing compound, did not stimulate sexual reproduction. In addition, an ACC analog, 1-aminocyclobutane-1-carboxylic acid (ACBC), which contains a four membered carbocyclic ring, promoted sexual reproduction and enhanced tolerance to oxidative stress in the same manner as ACC, but 1-aminocyclopentane-1-carboxylic acid (cycloleucine; which contains a cyclopentane ring) did not. The application of ACC increased the generation of reactive oxygen species (ROS) and induced the expression of PyRboh gene encoding NADPH oxidase. ACC also stimulated the synthesis of ascorbate (AsA) by inducing transcripts of PyGalLDH, which encodes galactono-1,4-lactone dehydrogenase, the catalyst for the final enzymatic step of the AsA biosynthetic pathway. Conversely, ACC caused a decrease in the synthesis of glutathione (GSH) by repressing transcripts of PyGCL, which encodes glutamate cysteine ligase, the catalyst for the rate-limiting step in the formation of GSH. These results suggest a possible role played by ACC as a signaling molecule independent from ethylene in the regulation of sexual reproduction through alterations to the redox state in P. yezoensis.

Characterization and expression profiles of small heat shock proteins in the marine red alga Pyropia yezoensis.

Small heat shock proteins (sHSPs) are found in all three domains of life (Bacteria, Archaea, and Eukarya) and play a critical role in protecting organisms from a range of environmental stresses. However, little is known about their physiological functions in red algae. Therefore, we characterized the sHSPs (PysHSPs) in the red macroalga Pyropia yezoensis, which inhabits the upper intertidal zone where it experiences fluctuating stressful environmental conditions on a daily and seasonal basis, and examined their expression profiles at different developmental stages and under varying environmental conditions. We identified five PysHSPs (PysHSP18.8, 19.1, 19.2, 19.5, and 25.8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that expression of the genes PysHSP18.8, PysHSP19.5, and PysHSP25.8 was repressed at all the developmental stages under normal conditions, whereas PysHSP19.1 and PysHSP19.2 were overexpressed in mature gametophytes and sporophytes. Exposure of the gametophytes to high temperature, oxidative stress, or copper significantly increased the mRNA transcript levels of all the five genes, while exogenous application of the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) significantly increased the expression levels of PysHSP19.2, PysHSP19.5, and PysHSP25.8. These findings will help to further our understanding of the role of PysHSP genes and provide clues about how Pyropia species can adapt to the stressful conditions encountered in the upper intertidal zone during their life cycle.

Complete sequence of mitochondrial DNA of Gloiopeltis furcata (Postels and Ruprecht) J. Agardh.

Although most red algae produce agar and carrageenan, Gloiopeltis furcata produces funoran as polysaccharide component. In this study, the complete G. furcata mitochondrial genome was determined. It had a circular mapping molecular with the length of 25,636 bp and contained 49 genes including 24 protein-coding, two rRNA, and 23 tRNA. Phylogenetic analysis showed that G. furcata was separated with the other polysaccharide-producing red algae. This is the first report of complete mitochondrial genome from funoran producing red algae.

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.

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 an Eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia yezoensis.

BACKGROUND: Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL-7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis. METHODS: The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated. RESULTS: PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35oC and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage. CONCLUSION: This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.

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.

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 ß-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.

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 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.

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 â-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.