Genome-Wide Identification and Analysis of MYB Transcription Factors in Pyropia yezoensis.

MYB transcription factors are one of the largest transcription factor families in plants, and they regulate numerous biological processes. Red algae are an important taxonomic group and have important roles in economics and research. However, no comprehensive analysis of the MYB gene family in any red algae, including Pyropia yezoensis, has been conducted. To identify the MYB gene members of Py. yezoensis, and to investigate their family structural features and expression profile characteristics, a study was conducted. In this study, 3 R2R3-MYBs and 13 MYB-related members were identified in Py. yezoensis. Phylogenetic analysis indicated that most red algae MYB genes could be clustered with green plants or Glaucophyta MYB genes, inferring their ancient origins. Synteny analysis indicated that 13 and 5 PyMYB genes were orthologous to Pyropia haitanensis and Porphyra umbilicalis, respectively. Most Bangiaceae MYB genes contain several Gly-rich motifs, which may be the result of an adaptation to carbon limitations and maintenance of important regulatory functions. An expression profile analysis showed that PyMYB genes exhibited diverse expression profiles. However, the expression patterns of different members appeared to be diverse, and PyMYB5 was upregulated in response to dehydration, low temperature, and Pythium porphyrae infection. This is the first comprehensive study of the MYB gene family in Py. Yezoensis and it provides vital insights into the functional divergence of MYB genes.

Metagenome-Assembled Genomes From Pyropia haitanensis Microbiome Provide Insights Into the Potential Metabolic Functions to the Seaweed.

Pyropia is an economically important edible red alga worldwide. The aquaculture industry and Pyropia production have grown considerably in recent decades. Microbial communities inhabit the algal surface and produce a variety of compounds that can influence host adaptation. Previous studies on the Pyropia microbiome were focused on the microbial components or the function of specific microbial lineages, which frequently exclude metabolic information and contained only a small fraction of the overall community. Here, we performed a genome-centric analysis to study the metabolic potential of the Pyropia haitanensis phycosphere bacteria. We reconstructed 202 unique metagenome-assembled genomes (MAGs) comprising all major taxa present within the P. haitanensis microbiome. The addition of MAGs to the genome tree containing all publicly available Pyropia-associated microorganisms increased the phylogenetic diversity by 50% within the bacteria. Metabolic reconstruction of the MAGs showed functional redundancy across taxa for pathways including nitrate reduction, taurine metabolism, organophosphorus, and 1-aminocyclopropane-1-carboxylate degradation, auxin, and vitamin B12 synthesis. Some microbial functions, such as auxin and vitamin B12 synthesis, that were previously assigned to a few Pyropia-associated microorganisms were distributed across the diverse epiphytic taxa. Other metabolic pathways, such as ammonia oxidation, denitrification, and sulfide oxidation, were confined to specific keystone taxa.

Comparative Gene Expression and Physiological Analyses Reveal Molecular Mechanisms in Wound-Induced Spore Formation in the Edible Seaweed Nori.

Genetic reprogramming of differentiated cells is studied broadly in multicellular Viridiplantae as an adaptation to herbivory or damage; however, mechanisms underlying cell development and redifferentiation are largely unknown in red algae, their nearest multicellular relatives. Here we investgate cell reprogramming in the widely cultivated, edible seaweed Neopyropia yezoesis ("nori"), where vegetative cells in wounded blades differentiate and release as large numbers of asexual spores. Based upon physiological changes and transcriptomic dynamics after wound stress in N. yezoensis and its congener Neoporphyra haitanensis, another cultivar that does not differentiate spores after wounding, we propose a three-phase model of wound-induced spore development in N. yezoensis. In Phase I, propagation of ROS by RBOH and SOD elicites systematic transduction of the wound signal, while Ca2+ dependent signaling induces cell reprogramming. In Phase II, a TOR signaling pathway and regulation of cyclin and CDK genes result in cell divisions that spread inward from the wound edge. Once sporangia form, Phase III involves expression of proteins required for spore maturation and cell wall softening. Our analyses not only provide the first model for core molecular processes controlling cellular reprogramming in rhodophytes, but also have practical implications for achieving greater control over seeding in commercial nori farming.

Genome-wide analysis of HSP70 gene superfamily in Pyropia yezoensis (Bangiales, Rhodophyta): identification, characterization and expression profiles in response to dehydration stress.

BACKGROUND: Heat shock proteins (HSPs) perform a fundamental role in protecting plants against abiotic stresses. Individual family members have been analyzed in previous studies, but there has not yet been a comprehensive analysis of the HSP70 gene family in Pyropia yezoensis. RESULTS: We investigated 15 putative HSP70 genes in Py. yezoensis. These genes were classified into two sub-families, denoted as DnaK and Hsp110. In each sub-family, there was relative conservation of the gene structure and motif. Synteny-based analysis indicated that seven and three PyyHSP70 genes were orthologous to HSP70 genes in Pyropia haitanensis and Porphyra umbilicalis, respectively. Most PyyHSP70s showed up-regulated expression under different degrees of dehydration stress. PyyHSP70-1 and PyyHSP70-3 were expressed in higher degrees compared with other PyyHSP70s in dehydration treatments, and then expression degrees somewhat decreased in rehydration treatment. Subcellular localization showed PyyHSP70-1-GFP and PyyHSP70-3-GFP were in the cytoplasm and nucleus/cytoplasm, respectively. Similar expression patterns of paired orthologs in Py. yezoensis and Py. haitanensis suggest important roles for HSP70s in intertidal environmental adaptation during evolution. CONCLUSIONS: These findings provide insight into the evolution and modification of the PyyHSP70 gene family and will help to determine the functions of the HSP70 genes in Py. yezoensis growth and development.

Structural and Functional Impacts of Microbiota on Pyropia yezoensis and Surrounding Seawater in Cultivation Farms along Coastal Areas of the Yellow Sea.

Pyropia yezoensis is the most important commercial edible red algae in China, carrying a variety of resident microbes at its surface. To understand microbiome diversity, community structure, interactions and functions with hosts in this regard, thalli and seawater sampleswere collected from Yantai and Rizhao cultivation farms in the Yellow Sea. The thalli and seawater samples (n = 12) were collected and studied using an Illumina NovaSeq 6000 platform and 16S ribosomal RNA (rRNA) gene sequencing, along with the consideration of environmental factors. Bacterial communities in association with P. yezoensis and surrounding seawater were predominated by Cyanobacteria, Proteobacteria, and Bacteroidetes. The variability of bacterial communities related to P. yezoensis and seawater were predominantly shaped by nitrate (NO3), ammonium (NH4), and temperature. Cluster analysis revealed a close relationship between thalli (RTH and YTH) and seawater (RSW and YSW) in terms of the residing bacterial communities, respectively. PICRUSt analysis revealed the presence of genes associated with amino acid transportation and metabolism, which explained the bacterial dependence on algal-provided nutrients. This study reveals that the diversity of microbiota for P. yezoensis is greatly influenced by abiotic factors and algal organic exudates which trigger chemical signaling and transportation responses from the bacterial community, which in turn activates genes to metabolize subsequent substrates.

Pyropia yezoensis genome reveals diverse mechanisms of carbon acquisition in the intertidal environment.

Changes in atmospheric CO2 concentration have played a central role in algal and plant adaptation and evolution. The commercially important red algal genus, Pyropia (Bangiales) appears to have responded to inorganic carbon (Ci) availability by evolving alternating heteromorphic generations that occupy distinct habitats. The leafy gametophyte inhabits the intertidal zone that undergoes frequent emersion, whereas the sporophyte conchocelis bores into mollusk shells. Here, we analyze a high-quality genome assembly of Pyropia yezoensis to elucidate the interplay between Ci availability and life cycle evolution. We find horizontal gene transfers from bacteria and expansion of gene families (e.g. carbonic anhydrase, anti-oxidative related genes), many of which show gametophyte-specific expression or significant up-regulation in gametophyte in response to dehydration. In conchocelis, the release of HCO3- from shell promoted by carbonic anhydrase provides a source of Ci. This hypothesis is supported by the incorporation of 13C isotope by conchocelis when co-cultured with 13C-labeled CaCO3.

Fine Mapping to Identify the Functional Genetic Locus for Red Coloration in Pyropia yezoensis Thallus.

Pyropia yezoensis, commonly known as "Nori" or "Laver" is an economically important marine crop. In natural or selected populations of P. yezoensis, coloration mutants are frequently observed. Various coloration mutants are excellent materials for genetic research and study photosynthesis. However, the candidate gene controlling the Pyropia coloration phenotype remains unclear to date. QTL-seq, in combination with kompetitive allele-specific PCR (KASP) and RNA-seq, can be generally applied to population genomics studies to rapidly identify genes that are responsible for phenotypes showing extremely opposite traits. Through cross experiments between the wild line RZ and red-mutant HT, offsprings with 1-4 sectors chimeric blade were generated. Statistical analyses revealed that the red thallus coloration phenotype is conferred by a single nuclear allele. Two-pair populations, consisting of 24 and 56 wild-type/red-type single-genotype sectors from F1 progeny, were used in QTL-seq to detect a genomic region in P. yezoensis harboring the red coloration locus. Based on a high-quality genome, we first identified the candidate region within a 3.30-Mb region at the end of chromosome 1. Linkage map-based QTL analysis was used to confirm the candidate region identified by QTL-seq. Then, four KASP markers developed in this region were used to narrow down the candidate region to a 1.42-Mb region. Finally, we conducted RNA-seq to focus on 13 differentially expressed genes and further predicted rcl-1, which contains one non-synonymous SNP [A/C] in the coding region that could be regulating red thallus coloration in P. yezoensis. Our results provide novel insights into the underlying mechanism controlling blade coloration, which is a desirable trait in algae.

Comparative Quantitative Proteomics Reveals the Desiccation Stress Responses of the Intertidal Seaweed NEOPORPHYRA haitanensis.

Neoporphyra haitanensis is an economically important red seaweed that inhabits upper intertidal zones. The thallus tolerates extreme fluctuating environmental stresses (e.g., surviving more than 80% water loss during low tides). To elucidate the global molecular responses relevant to this outstanding desiccation tolerance, a quantitative proteomics analysis of N. haitanensis under different desiccation treatments as well as rehydration was performed. According to the clustering of expression patterns and the functional interpretation of the 483 significantly differentially expressed proteins, a three-stage cellular response to desiccation stress and subsequent rehydration was proposed. Stage I: at the beginning of water loss, multiple signal transduction pathways were triggered including lipid signaling, protein phosphorylation cascades, and histone acetylation controlling acetate biosynthesis to further modulate downstream hormone signaling. Protein protection by peptidyl-prolyl isomerase and ROS scavenging systems were also immediately switched on. Stage II: with the aggravation of stress, increases in antioxidant systems, the accumulation of LEA proteins, and the temporary biosynthesis of branched starch were observed. Multiple enzymes involved in redox homeostasis, including peroxiredoxin, thioredoxin, ascorbate peroxidase, superoxide dismutase, glutathione peroxidase, and glutathione reductase, were hypothesized to function in specific cellular compartments. Stage III: when the desiccated thalli had rehydrated for 30 mins, photosynthesis and carbon fixation were recovered, and antioxidant activities and protein structure protection were maintained at a high level. This work increases the understanding of the molecular responses to environmental stresses via a proteomic approach in red seaweeds and paves the way for further functional studies and genetic engineering.

A chromosome-level genome assembly of Pyropia haitanensis (Bangiales, Rhodophyta).

Pyropia haitanensis (Bangiales, Rhodophyta), a major economically important marine crop, is also considered as an ideal research model of Rhodophyta to address several major biological questions such as sexual reproduction and adaptation to intertidal abiotic stresses. However, comparative genomic analysis to decipher the underlying molecular mechanisms is hindered by the lack of high-quality genome information. Therefore, we integrated sequencing data from Illumina short-read sequencing, PacBio single-molecule sequencing and BioNano optical genome mapping. The assembled genome was approximately 53.3 Mb with an average GC% of 67.9%. The contig N50 and scaffold N50 were 510.3 kb and 5.8 Mb, respectively. Additionally, 10 superscaffolds representing 80.9% of the total assembly (42.7 Mb) were anchored and orientated to the 5 linkage groups based on markers and genetic distance; this outcome is consistent with the karyotype of five chromosomes (n = 5) based on cytological observation in P. haitanensis. Approximately 9.6% and 14.6% of the genomic region were interspersed repeat and tandem repeat elements, respectively. Based on full-length transcriptome data generated by PacBio, 10,903 protein-coding genes were identified. The construction of a genome-wide phylogenetic tree demonstrated that the divergence time of P. haitanensis and Porphyra umbilicalis was ~204.4 Ma. Interspecies comparison revealed that 493 gene families were expanded and that 449 were contracted in the P. haitanensis genome compared with those in the Po. umbilicalis genome. The genome identified is of great value for further research on the genome evolution of red algae and genetic adaptation to intertidal stresses.

Transcriptomic Insights into Innate Immunity Responding to Red Rot Disease in Red Alga Pyropia yezoensis.

Pyropia yezoensis, one of the most economically important marine algae, suffers from the biotic stress of the oomycete necrotrophic pathogen Pythium porphyrae. However, little is known about the molecular defensive mechanisms employed by Pyr. yezoensis during the infection process. In the present study, we defined three stages of red rot disease based on histopathological features and photosynthetic physiology. Transcriptomic analysis was carried out at different stages of infection to identify the genes related to the innate immune system in Pyr. yezoensis. In total, 2139 up-regulated genes and 1672 down-regulated genes were identified from all the infected groups. Pathogen receptor genes, including three lectin genes (pattern recognition receptors (PRRs)) and five genes encoding typical plant R protein domains (leucine rich repeat (LRR), nucleotide binding site (NBS), or Toll/interleukin-1 receptor (TIR)), were found to be up-regulated after infection. Several defense mechanisms that were typically regarded as PAMP-triggered immunity (PTI) in plants were induced during the infection. These included defensive and protective enzymes, heat shock proteins, secondary metabolites, cellulase, and protease inhibitors. As a part of the effector-triggered immunity (ETI), the expression of genes related to the ubiquitin-proteasome system (UPS) and hypersensitive cell death response (HR) increased significantly during the infection. The current study suggests that, similar to plants, Pyr. yezoensis possesses a conserved innate immune system that counters the invasion of necrotrophic pathogen Pyt. porphyrae. However, the innate immunity genes of Pyr. yezoensis appear to be more ancient in origin compared to those in higher plants.

The complete plastid genome of a marine microalgae Cryptophyceae sp. CCMP2293 (Cryptophyta).

In this study, we present the complete plastid genome of Cryptophyceae sp. CCMP2293. The circular genome is 139,208 bp in length and contains 142 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes, 6 ribosome RNA (rRNA) genes, and 1 transfer-messenger RNA (tmRNA) gene. The overall nucleotide composition is: 33.6% A, 32.5% T, 16.8% C, and 17.1% G with a total A + T content of 66.1%. The phylogenetic tree was constructed to explore the taxonomic status of Cryptophyceae sp. CCMP2293, which is closely related to G. theta and R. salina.

Gene expression profiles of Pyropia yezoensis in response to dehydration and rehydration stresses.

Pyropia yezoensis is an economically important marine macroalgae, naturally distributed in the upper intertidal zone. Owing to the nature of its habitat, the thallus will periodically be exposed to seawater or atmosphere, and can lose up to 95% of its cellular water content. This makes the alga an ideal research model to investigate the mechanisms of desiccation tolerance. In this study, we investigated the response mechanisms to dehydration and rehydration stresses at the transcription level in Pyropia yezoensis. The differently expressed genes were analyzed based on the different functions of encoding proteins: effector proteins (chloroplast proteins, macromolecular protective substances, and toxicity degradation enzymes) and regulatory proteins (protein kinases and phosphatases). Under osmotic stress, the unigenes related to photosynthesis were down-regulated significantly while those encoding glutathione transferase, superoxide dismutase and heat shock proteins were up-regulated significantly. We inferred that the photosynthetic activity was reduced to prevent damage caused by photosynthetic by-products and that the expression of antioxidant enzyme was increased to prevent the damage associated with reactive oxygen species. Additionally, unigenes encoding serine/threonine kinases and phospholipases were up-regulated in response to osmotic stress, indicating that these kinases play an important role in osmotolerance. Our work will serve as an essential foundation for the understanding of desiccation tolerance mechanisms in Pyropia yezoensis in the upper intertidal zones of rocky coasts.

A comparative study of the photosynthetic capacity in two green tide macroalgae using chlorophyll fluorescence.

Green tides have occurred in the Yellow Sea, China, every year from 2007 to 2015. The free-floating Ulva prolifera (Müller) J. Agardh was the causative macroalgal species. The co-occurring, attached U. intestinalis was also observed. Photosynthetic capacities were determined using chlorophyll fluorescence in situ and after 7 days lab acclimation, and a significant differences were noted. Pigment composition showed no obvious differences, but concentrations varied significantly, especially chlorophyll b in U. prolifera two times increase was observed after acclimation. The optimal photochemical efficiency of PS II (Fv/Fm) was significantly higher in U. prolifera. Photosynthetic rate (α), maximum relative electron transport rate (rETRmax), and minimum saturating irradiance (Ek), obtained from rapid light response curves (RLCs), showed almost the same photosynthetic physiological status as Fv/Fm. Quenching coefficients and low temperature (77 K) chlorophyll fluorescence emission spectra of thylakoid membranes analysis showed U. prolifera has a better recovery activity and plasticity of PSII than U. intestinalis. Furthermore, energy dissipation via non-photochemical quenching (NPQ) and state transitions showed efficacious photoprotection solution especially in U. prolifera suffered from the severe stresses. Results in the present study suggested that U. prolifera's higher photosynthetic capacity would contribute to its free-floating proliferation, and efficacious photoprotection in addition to favorable oceanographic conditions and high nutrient levels support its growth and aggregation.

The complete chloroplast genome of Guillardia theta strain CCMP2712.

The complete chloroplast sequence of Cryptophyceae algae Guillardia theta strain CCMP2712 was determined in this study. The genome consists of 138 455 bp containing a pair of rRNA-coding invert repeats of 4973 bp. The overall GC contents of the chloroplast genome were 32.9%. A total of 159 functional genes were annotated, which included 124 protein-coding genes, 30 tRNAs, 1tmRNA and 4 rRNA genes. Short intergenic regions, no introns and some overlapping genes make this cp genome compact. Phylogenetic analysis with the related algae cp genomes revealed that G. theta strain CCMP2712 a typical Cryptophyta algae and had a close genetic relationship with Rhodomonas salina (EF508371) and another previously reported G. theta (AF041468).

Complete mitochondrial genome of Fistulifera solaris (Bacillariophycidae).

The complete mitochondrial DNA of pennate diatom Fistulifera solaris was determined. The circular mitochondrial genome is 39 648 bp in length with 28.1% GC content and contains 63 genes, including 33 protein-coding genes, three conserved open reading frame (ORF154, ORF192 and ORF251), 25 tRNA and 2 rRNA genes. All protein-coding genes have AUG as start codon. A group I intron was found in nad11 gene. Both the gene content and the structure of Fistulifera solaris mitochondrial genome are very similar to Berkeleya fennica mitogenome (KM886611). Phylogeny analysis indicate F. solaris a close genetic relationship with Berkeleya fennica. This new mitogenome will provide more useful information for exploration in diatoms diversity and evolution.

Elucidation of matrix effects and performance of solid-phase extraction for LC-MS/MS analysis of ß-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) neurotoxins in cyanobacteria.

A liquid chromatography-mass spectrometry (LC-MS/MS) method using hydrophilic interaction liquid chromatography (HILIC) was developed for the analysis of neurotoxins ß-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), using multiple reaction monitoring (MRM) scan mode. Oasis-MCX and Strata-X-C polymeric cation-exchange cartridges were used to clean extracts of cyanobacterial cultures, including two strains of Microcystis aeruginosa and one strain of Nostoc sp. The performance of the solid-phase extraction (SPE) cartridges for BMAA and DAB were evaluated using mixed standards and spiked cyanobacterial extracts, which demonstrated recoveries of BMAA and DAB ranging from 66% to 91%. Matrix effects in LC-MS/MS were evaluated, and while there was no effect on BMAA quantitation, suppression of DAB was found. Full scan (Q1) and enhanced product ion (EPI) monitoring showed that the DAB suppression may be due to closely eluting compounds, including lysine, histidine, arginine and three other compounds with [M + H](+) m/z of 88, 164 and 191. The procedures developed allow the sensitive and effective analysis of trace BMAA and DAB levels in cyanobacteria. While DAB was confirmed to be present, no BMAA was found in the cyanobacterial samples tested in the present study.