Environmental heterogeneity caused by large-scale cultivation of Pyropia haitanensis shapes multi-group biodiversity distribution in coastal areas.

The response of marine biodiversity to mariculture has long been a research focus in marine ecology. However, the effects of seaweed cultivation on biological community assembly are poorly understood, especially in diverse communities with distinct ecological characteristics. In this study, we used environmental DNA metabarcoding to investigate the spatial distribution patterns of bacterial, protistan, and metazoan diversity, aiming to reveal the mechanisms of community assembly in the Pyropia haitanensis cultivation zone along the Fujian coast, China. We found that, compared with the biological communities in control zones, those in P. haitanensis cultivation zones exhibited stronger geographic distance-decay patterns and displayed more complex and stable network structures. Deterministic processes (environmental selection) played a more important role in the assembly of bacterial, protistan, and metazoan communities in P. haitanensis cultivation zones, especially metazoan communities. Variance partitioning analysis showed that environmental variables made greater contributions to the diversity of the three types of communities within the P. haitanensis cultivation zones than in the control zones. Partial least squares path modeling analysis identified nitrate­nitrogen (NO3-N), pH, particulate organic carbon (POC), and dissolved organic carbon (DOC) as the key environmental variables affecting biodiversity. Overall, the environmental heterogeneity caused by the large-scale cultivation of P. haitanensis could be the crucial factor influencing the composition and structure of various biological communities. Our results highlight the importance of the responses of multi-group organisms to the cultivation of seaweed, and provide insights into the coexistence patterns of biodiversity at the spatial scale.

PhbZIP2 regulates photosynthesis-related genes in an intertidal macroalgae, Pyropia haitanensis, under stress.

Intertidal macroalgae are important research subjects in stress biology. Basic region-leucine zipper transcription factors (bZIPs) play an important regulatory role in the expression of target genes under abiotic stress. We herein identified a bZIP2 gene PhbZIP2 to regulate abiotic stress tolerance in Pyropia haitanensis, a representative intertidal macroalgal species. Cloning and sequencing of the cDNA characterized a BRLZ structure and an α coiled-coil structure between amino acids and Expression of PhbZIP2 was detected to upregulate under both high temperature and salt stresses. A DAP-seq analysis revealed the PhbZIP2-binding motifs of (T/C)TCCA(C/G) and A (A/G)AAA (G/A), which differed from the conserved motifs in plants. Overexpression of PhbZIP2 was indicative of a high temperature and salt stress tolerances in transgenic Chlamydomonas reinhardtii. It was suggested that PhbZIP2 was probably involved in regulating expression of the photosynthetic-related genes and the response to the abiotic stresses in P. haitanensis, which provide new insights for elucidating efficient adaptation strategies of intertidal macroalgae.

Interactions between nitrogen and phosphorus modulate the food quality of the marine crop Pyropia haitanensis (T. J. Chang & B. F. Zheng) N. Kikuchi & M. Miyata (Porphyra haitanensis).

The quality of Pyropia haitanensis (T. J. Chang & B. F. Zheng) N. Kikuchi & M. Miyata (Porphyra haitanensis) is directly affected by nutrient availability. However, the molecular mechanism underlying the synergistic regulatory effects of nitrogen (N) and phosphorus (P) availability on P. haitanensis quality is unknown. Here, we performed physiological and multi-omics analyses to reveal the combined effects of N and P on P. haitanensis quality. The pigments accumulated under high N because of increases in N metabolism and porphyrin metabolism, ultimately resulting in intensely colored thalli. High N also promoted amino acid metabolism and inosine 5'-mononucleotide (IMP) synthesis, but inhibited carbohydrates accumulation. This resulted in increased amino acid, IMP and decreased agaro-carrageenan and cellulose contents, thereby improving the nutritional value and taste. Furthermore, high P promoted carbon metabolism and amino acid metabolism.This study provided the basis for elucidating the mechanism behind N and P regulating the seaweed quality.

Investigating the Mechanisms Underlying the Low Irradiance-Tolerance of the Economically Important Seaweed Species Pyropia haitanensis.

Pyropia haitanensis, one of the most economically and ecologically important seaweed species, is often exposed to persistent or transient low irradiance (LI), resulting in limited yield and quality. However, the mechanisms mediating P. haitanensis responses to LI are largely unknown. In this study, LI-tolerant (LIT) and LI-sensitive (LIS) P. haitanensis strains were compared regarding their physiological and transcriptomic changes induced by 1 and 4 days of LI (5 µmol photons/m2·s). The results indicated that the inhibition of photomorphogenesis and decreases in photosynthesis and photosynthetic carbon fixation as the duration of LI increased are the key reasons for retarded blade growth under LI conditions. A potential self-amplifying loop involving calcium signaling, phosphatidylinositol signaling, reactive oxygen species signaling, and MAPK signaling may be triggered in blades in response to LI stress. These signaling pathways might activate various downstream responses, including improving light energy use, maintaining cell membrane stability, mitigating oxidative damage, to resist LI stress. Additionally, the LIT strain maintained transcriptional homeostasis better than the LIS strain under LI stress. Specifically, photosynthesis and energy production were relatively stable in the LIT strain, which may help to explain why the LIT strain was more tolerant to LI stress than the LIS strain. The findings of this study provide the basis for future investigations on the precise mechanisms underlying the LI stress tolerance of P. haitanensis.

Cultivation of different seaweed species and seasonal changes cause divergence of the microbial community in coastal seawaters.

Although the effects of certain species of seaweed on the microbial community structure have long been a research focus in marine ecology, the response of the microbial community to seasons and different seaweed species is poorly understood. In the present study, a total of 39 seawater samples were collected during 3 months from three zones: Neoporphyra haitanensis cultivation zones (P), Gracilaria lemaneiformis-Saccharina japonica mixed cultivation zones (G), and control zones (C). These samples were then analyzed using 18S and 16S rRNA gene sequencing to ascertain the fungal and bacterial communities, respectively, along with the determination of environmental factors. Our results showed that increased dissolved oxygen (DO), decreased inorganic nutrients, and released dissolved organic matter (DOM) in seaweed cultivation zone predominantly altered the variability of eukaryotic and prokaryotic microbial communities. Certain microbial groups such as Aurantivirga, Pseudomonas, and Woeseia were stimulated and enriched in response to seaweed cultivation, and the enriched microorganisms varied across seaweed cultivation zones due to differences in the composition of released DOM. In addition, seasonal changes in salinity and temperature were strongly correlated with microbial community composition and structure. Our study provides new insights into the interactions between seaweed and microbial communities.

Na + /K + -ATPase regulates the K + /Na + homeostasis in the intertidal macroalgae, Neoporphyra haitanensis, in response to salt stress.

In plants under hypersaline stress, the main transporter that extrudes sodium ions (Na + ) is the Na + /H + antiporter SOS1. Different from land plants, the intertidal macroalgae, Neopyropia/Neoporphyra contains an animal-type Na + /K + -ATPase as well as the SOS1 system. However, the contribution of Na + /K + -ATPase to the K + /Na + homeostasis of intertidal macroalgae remains unclear. In this study, we analyzed the function of Na + /K + -ATPase in the response of Neoporphyra haitanensis to salt stress from the perspective of ion transport dynamics. Both the transcript level of NhNKA2 and enzyme activity of Na + /K + -ATPase increased in the early response of N. haitanensis thalli to hypersaline stress. Addition of ouabain, an inhibitor of Na + /K + -ATPase, resulted in Na + accumulation in the cells, severe K + leakage from the thalli, and then remarkably disturbed the K + /Na + homeostasis in N. haitanensis thalli. This disruption might induce a significant decrease in photosynthesis and a severe oxidative damage in thalli. Accordingly, these results suggested that the important role of Na + /K + -ATPase in the resistance of intertidal macroalgae to hypersaline stress, and shed light on the diversity of K + /Na + homeostasis maintenance mechanisms in plants.

Transcriptome Co-expression Network Analysis Identifies Key Genes Regulating Conchosporangia Maturation of Pyropia haitanensis.

Conchosporangia maturation is crucial for the yield of Pyropia/Porphyra. However, the molecular mechanisms underlying this process are poorly understood. In this study, we selected two strains of Pyropia haitanensis that show significant differences in conchosporangia maturation as materials to produce RNA-Seq libraries. Then, we identified key molecular pathways and genes involved in conchosporangia maturation by conducting a weighted gene co-expression network analysis. Two specific modules were identified, and included functions such as phosphorus metabolism, lipid metabolism, and the phosphatidylinositol signaling system. The hub genes that responded positively during conchosporangia maturation encoded diacylglycerol kinase (DGK) and phosphatidylinositol-3-phosphate-5-kinase, which are involved in the synthesis of phosphatidic acid, a key component of lipid metabolism. A full-length DGK sequence of P. haitanensis, designated as PhDGK1, was obtained by rapid-amplification of cDNA ends. Conserved motif and phylogenetic tree analyses showed that PhDGK1 belongs to DGK Cluster II. The transcript level of PhDGK1 increased during conchosporangia maturation in both strains, but increased earlier, and to higher levels, in the early-maturing strain than in the late-maturing strain. This pattern of gene expression was consistent with the patterns of maturity and changes in pigment contents. These results indicate that lipid metabolism plays a key role in regulating conchosporangia maturation in Pyropia spp., and that PhDGK1 might be a useful molecular marker for breeding new early-maturing strains.

The response of Pyropia haitanensis to inorganic arsenic under laboratory culture.

Up to now, complicated organoarsenicals were mainly identified in marine organisms, suggesting that these organisms play a critical role in arsenic biogeochemical cycling because of low phosphate and relatively high arsenic concentration in the marine environment. However, the response of marine macroalgae to inorganic arsenic remains unknown. In this study, Pyropia haitanensis were exposed to arsenate [As(V)] (0.1, 1, 10, 100 µM) or arsenite [As(III)] (0.1, 1, 10 µM) under laboratory conditions for 3 d. The species of water-soluble arsenic, the total concentration of lipid-soluble and cell residue arsenic of the algae cells was analyzed. As(V) was mainly transformed into oxo-arsenosugar-phosphate, with other arsenic compounds such as monomethylated, As(III), demethylated arsenic and oxo-arsenosugar-glycerol being likely the intermediates of arsenosugar synthesis. When high concentration of As(III) was toxic to P. haitanensis, As(III) entered into the cells and was transformed into less toxic organoarsenicals and As(V). Transcriptome results showed genes involved in DNA replication, mismatch repair, base excision repair, and nucleotide excision repair were up-regulated in the algae cells exposed to 10 µM As(V), and multiple genes involved in glutathione metabolism and photosynthetic were up-regulated by 1 µM As(III). A large number of ABC transporters were down-regulated by As(V) while ten genes related to ABC transporters were up-regulated by As(III), indicating that ABC transporters were involved in transporting As(III) to vacuoles in algae cells. These results indicated that P. haitanensis detoxifies inorganic arsenic via transforming them into organoarsenicals and enhancing the isolation of highly toxic As(III) in vacuoles.

Insight into transketolase of Pyropia haitanensis under desiccation stress based on integrative analysis of omics and transformation.

BACKGROUND: Pyropia haitanensis, distributes in the intertidal zone, can tolerate water losses exceeding 90%. However, the mechanisms enabling P. haitanensis to survive harsh conditions remain uncharacterized. To elucidate the mechanism underlying P. haitanensis desiccation tolerance, we completed an integrated analysis of its transcriptome and proteome as well as transgenic Chlamydomonas reinhardtii carrying a P. haitanensis gene. RESULTS: P. haitanensis rapidly adjusted its physiological activities to compensate for water losses up to 60%, after which, photosynthesis, antioxidant systems, chaperones, and cytoskeleton were activated to response to severe desiccation stress. The integrative analysis suggested that transketolase (TKL) was affected by all desiccation treatments. Transgenic C. reinhardtii cells overexpressed PhTKL grew better than the wild-type cells in response to osmotic stress. CONCLUSION: P. haitanensis quickly establishes acclimatory homeostasis regarding its transcriptome and proteome to ensure its thalli can recover after being rehydrated. Additionally, PhTKL is vital for P. haitanensis desiccation tolerance. The present data may provide new insights for the breeding of algae and plants exhibiting enhanced desiccation tolerance.

Regulatory mechanisms underlying the maintenance of homeostasis in Pyropia haitanensis under hypersaline stress conditions.

Intertidal macroalgae are highly resistant to hypersaline stress conditions. However, the underlying mechanism remains unknown. In the present study, the mechanism behind Pyropia haitanensis responses to two hypersaline stress conditions [100‰ (HSS_100) and 110‰ (HSS_110)] was investigated via analyses of physiological and transcriptomic changes. We observed that the differences between the responses of Py. haitanensis to HSS_100 and HSS_110 conditions involved the following three aspects: osmotic regulation, ionic homeostasis, and adjustment to secondary stresses. First, the water retention of Py. haitanensis was maintained through increased expansin production under HSS_100 conditions, while cell wall pectin needed to be protected from hydrolysis via the increased abundance of a pectin methylesterase inhibitor under HSS_110 conditions. Meanwhile, Py. haitanensis achieved stable and rapid osmotic adjustments because of the coordinated accumulation of inorganic ions (K+, Na+, and Cl-) and organic osmolytes (glycine betaine and trehalose) under HSS_100 conditions, but not under HSS_110 conditions. Second, Py. haitanensis maintained a higher K+/Na+ ratio under HSS_100 conditions than under HSS_110 conditions, mainly via the export of Na+ into the apoplast rather than compartmentalizing it into the vacuoles, and the enhanced uptake and retention of K+. However, K+/Na+ homeostasis was not completely disrupted during a short-term exposure to HSS_110 conditions. Finally, the Py. haitanensis antioxidant system scavenged more ROS and synthesized more heat shock proteins under HSS_100 conditions than under HSS_110 conditions, although thalli may have been able to maintain a certain redox balance during a short-term exposure to HSS_110 conditions. These differences may explain why Py. haitanensis can adapt to HSS_100 conditions rather than HSS_110 conditions, and also why the thalli exposed to HSS_110 conditions can recover after being transferred to normal seawater. Thus, the data presented herein may elucidate the mechanisms enabling Pyropia species to tolerate the sudden and periodic changes in salinity typical of intertidal systems.

Transcriptomic study to understand thermal adaptation in a high temperature-tolerant strain of Pyropia haitanensis.

Pyropia haitanensis, a high-yield commercial seaweed in China, is currently undergoing increasing levels of high-temperature stress due to gradual global warming. The mechanisms of plant responses to high temperature stress vary with not only plant type but also the degree and duration of high temperature. To understand the mechanism underlying thermal tolerance in P. haitanensis, gene expression and regulation in response to short- and long-term temperature stresses (SHS and LHS) was investigated by performing genome-wide high-throughput transcriptomic sequencing for a high temperature tolerant strain (HTT). A total of 14,164 differential expression genes were identified to be high temperature-responsive in at least one time point by high-temperature treatment, representing 41.10% of the total number of unigenes. The present data indicated a decrease in the photosynthetic and energy metabolic rates in HTT to reduce unnecessary energy consumption, which in turn facilitated in the rapid establishment of acclimatory homeostasis in its transcriptome during SHS. On the other hand, an increase in energy consumption and antioxidant substance activity was observed with LHS, which apparently facilitates in the development of resistance against severe oxidative stress. Meanwhile, ubiquitin-mediated proteolysis, brassinosteroids, and heat shock proteins also play a vital role in HTT. The effects of SHS and LHS on the mechanism of HTT to resist heat stress were relatively different. The findings may facilitate further studies on gene discovery and the molecular mechanisms underlying high-temperature tolerance in P. haitanensis, as well as allow improvement of breeding schemes for high temperature-tolerant macroalgae that can resist global warming.

Cells tile a flat plane by controlling geometries during morphogenesis of Pyropia thalli.

BACKGROUND: Pyropia haitanensis thalli, which are made of a single layer of polygonal cells, are a perfect model for studying the morphogenesis of multi-celled organisms because their cell proliferation process is an excellent example of the manner in which cells control their geometry to create a two-dimensional plane. METHODS: Cellular geometries of thalli at different stages of growth revealed by light microscope analysis. RESULTS: This study showed the cell division transect the middle of the selected paired-sides to divide the cell into two equal portions, thus resulting in cell sides ≥4 and keeping the average number of cell sides at approximately six even as the thallus continued to grow, such that more than 90% of the cells in thalli longer than 0.08 cm had 5-7 sides. However, cell division could not fully explain the distributions of intracellular angles. Results showed that cell-division-associated fast reorientation of cell sides and cell divisions together caused 60% of the inner angles of cells from longer thalli to range from 100-140°. These results indicate that cells prefer to form regular polygons. CONCLUSIONS: This study suggests that appropriate cell-packing geometries maintained by cell division and reorientation of cell walls can keep the cells bordering each other closely, without gaps.

Differential Proteomic Analysis by iTRAQ Reveals the Mechanism of Pyropia haitanensis Responding to High Temperature Stress.

Global warming increases sea temperature and leads to high temperature stress, which affects the yield and quality of Pyropia haitanensis. To understand the molecular mechanisms underlying high temperature stress in a high temperature tolerance strain Z-61, the iTRAQ technique was employed to reveal the global proteomic response of Z-61 under different durations of high temperature stress. We identified 151 differentially expressed proteins and classified them into 11 functional categories. The 4 major categories of these are protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. These findings indicated that photosynthesis, protein synthesis, and secondary metabolism are inhibited by heat to limit damage to a repairable level. As time progresses, misfolded proteins and ROS accumulate and lead to the up-regulation of molecular chaperones, proteases, and antioxidant systems. Furthermore, to cope with cells injured by heat, PCD works to remove them. Additionally, sulfur assimilation and cytoskeletons play essential roles in maintaining cellular and redox homeostasis. These processes are based on signal transduction in the phosphoinositide pathway and multiple ways to supply energy. Conclusively, Z-61 establishes a new steady-state balance of metabolic processes and survives under higher temperature stress.

Construction of a dense genetic linkage map and mapping quantitative trait loci for economic traits of a doubled haploid population of Pyropia haitanensis (Bangiales, Rhodophyta).

BACKGROUND: Pyropia haitanensis is one of the most economically important mariculture crops in China. A high-density genetic map has not been published yet and quantitative trait locus (QTL) mapping has not been undertaken for P. haitanensis because of a lack of sufficient molecular markers. Specific length amplified fragment sequencing (SLAF-seq) was developed recently for large-scale, high resolution de novo marker discovery and genotyping. In this study, SLAF-seq was used to obtain mass length polymorphic markers to construct a high-density genetic map for P. haitanensis. RESULTS: In total, 120.33 Gb of data containing 75.21 M pair-end reads was obtained after sequencing. The average coverage for each SLAF marker was 75.50-fold in the male parent, 74.02-fold in the female parent, and 6.14-fold average in each double haploid individual. In total, 188,982 SLAFs were detected, of which 6731 were length polymorphic SLAFs that could be used to construct a genetic map. The final map included 4550 length polymorphic markers that were combined into 740 bins on five linkage groups, with a length of 874.33 cM and an average distance of 1.18 cM between adjacent bins. This map was used for QTL mapping to identify chromosomal regions associated with six economically important traits: frond length, width, thickness, fresh weight, growth rates of frond length and growth rates of fresh weight. Fifteen QTLs were identified for these traits. The value of phenotypic variance explained by an individual QTL ranged from 9.59 to 16.61 %, and the confidence interval of each QTL ranged from 0.97 cM to 16.51 cM. CONCLUSIONS: The first high-density genetic linkage map for P. haitanensis was constructed, and fifteen QTLs associated with six economically important traits were identified. The results of this study not only provide a platform for gene and QTL fine mapping, map-based gene isolation, and molecular breeding for P. haitanensis, but will also serve as a reference for positioning sequence scaffolds on a physical map and will assist in the process of assembling the P. haitanensis genome sequence. This will have a positive impact on breeding programs that aim to increase the production and quality of P. haitanensis in the future.

Characterization of the global transcriptome for Pyropia haitanensis (Bangiales, Rhodophyta) and development of cSSR markers.

BACKGROUND: Pyropia haitanensis is an economically important mariculture crop in China and is also valuable in life science research. However, the lack of genetic information of this organism hinders the understanding of the molecular mechanisms of specific traits. Thus, high-throughput sequencing is needed to generate a number of transcriptome sequences to be used for gene discovery and molecular marker development. RESULTS: In this study, high-throughput sequencing was used to analyze the global transcriptome of P. haitanensis. Approximately 103 million 90 bp paired-end reads were generated using an Illumina HiSeq 2000. De novo assembly with paired-end information yielded 24,575 unigenes with an average length of 645 bp. Based on sequence similarity searches with known proteins, a total of 16,377 (66.64%) genes were identified. Of these annotated unigenes, 5,471 and 9,168 unigenes were assigned to gene ontology and clusters of orthologous groups, respectively. Searching against the KEGG database indicated that 12,167 (49.51%) unigenes mapped to 124 KEGG pathways. Among the carbon fixation pathways, almost all the essential genes related to the C3- and C4-pathways for P. haitanensis were discovered. Significantly different expression levels of three key genes (Rubisco, PEPC and PEPCK) in different lifecycle stages of P. haitanensis indicated that the carbon fixation pathway in the conchocelis and thallus were different, and the C4-like pathway might play important roles in the conchocelis stage. In addition, 2,727 cSSRs loci were identified in the unigenes. Among them, trinucleotide SSRs were the dominant repeat motif (87.17%, 2,377) and GCC/CCG motifs were the most common repeats (60.07%, 1,638). High quality primers to 824 loci were designed and 100 primer pairs were randomly evaluated in six strains of P. haitanensis. Eighty-seven primer pairs successfully yielded amplicons. CONCLUSION: This study generated a large number of putative P. haitanensis transcript sequences, which can be used for novel gene discovery and gene expression profiling analyses under different physiological conditions. A number of the cSSR markers identified can be used for molecular markers and will facilitate marker assisted selection in P. haitanensis breeding. These sequences and markers will provide valuable resources for further P. haitanensis studies.