Isolation and functional verification of an aspartate aminotransferase gene from Neoporphyra haitanensis.

BACKGROUND: Neoporphyra haitanensis is a commercial laver species in China. Aspartic acid is an important flavor amino acid, and aspartate aminotransferase (AAT) is a crucial enzyme in its biosynthesis. In this study, we cloned one AAT gene (NhAAT) from the red alga N. haitanensis and investigated its sequence structure, transcriptional expression and enzymatic characteristics. The purpose of our research is to obtain a functional AAT responsible for the biosynthesis of aspartic acid from red seaweeds, which has the potential to influence the flavor of N. haitanensis. RESULTS: Sequence analysis showed that NhAAT contains a conserved domain of Aminotran_1_2, which belongs to the transaminase superfamily. The secondary structure of NhAAT is dominated by α-helix. The results of enzymatic characterization illustrated that the NhAAT has highest catalytic activity at 45 °C and pH 7.5 in both forward and reverse reactions. The calculated Km values of NhAAT was 5.67 and 6.16 mM for L-glutamic acid and L-aspartic acid, respectively. Quantitative analysis showed that the NhAAT expression of N. haitanensis collected in late harvest (Dec) was 4.5 times that of N. haitanensis collected in early harvest (Oct), while the aspartic acid content of N. haitanensis collected in late harvest (Dec) was 1.2 times that of N. haitanensis collected in early harvest (Oct). CONCLUSION: The results of enzyme kinetics indicated that NhAAT prefers to catalyze the reaction in the direction of aspartic acid production. Moreover, the trend of NhAAT expression level was consistent with that of aspartic acid content in N. haitanensis in different harvest periods. Our research is helpful to understand the accumulation and regulation of amino acids in N. haitanensis in different habitats and the taste difference of N. haitanensis in different harvest periods.

Novel Chitin Deacetylase from Thalassiosira weissflogii Highlights the Potential for Chitin Derivative Production.

ß-Chitin is an important carbon fixation product of diatoms, and is the most abundant nitrogen-containing polysaccharide in the ocean. It has potential for widespread application, but the characterization of chitin-related enzymes from ß-chitin producers has rarely been reported. In this study, a chitin deacetylase (TwCDA) was retrieved from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database and was heterologously expressed in vitro for functional analysis. The results showed that both the full-length sequence (TwCDA) and the N-terminal truncated sequence (TwCDA-S) had chitin deacetylase and chitinolytic activities after expression in Escherichia coli. High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) indicated that TwCDA and TwCDA-S could catalyze the deacetylation of oligosaccharide (GlcNAc)5. TwCDA had higher deacetylase activity, and also catalyzed the deacetylation of the ß-chitin polymer. A dinitrosalicylic acid (DNS) assay showed that TwCDA-S had high chitinolytic activity for (GlcNAc)5, and the optimal reaction temperature was 35 °C. Liquid chromatography combined with time-of-flight mass spectrometry (LC-coTOF-MS) detected the formation of a N-acetylglucosamine monomer (C8H15NO6) in the reaction mixture. Altogether, we isolated a chitin deacetylase from a marine diatom, which can catalyze the deacetylation and degradation of chitin and chitin oligosaccharides. The relevant results lay a foundation for the internal regulation mechanism of chitin metabolism in diatoms and provide a candidate enzyme for the green industrial preparation of chitosan and chitin oligosaccharides.

Characterization of a Marine Diatom Chitin Synthase Using a Combination of Meta-Omics, Genomics, and Heterologous Expression Approaches.

ß-Chitin has important ecological and physiological roles and potential for widespread applications, but the characterization of chitin-related enzymes from ß-chitin producers was rarely reported. Querying against the Tara Oceans Gene Atlas, 4,939 chitin-related unique sequences from 12 Pfam accessions were found in Bacillariophyta metatranscriptomes. Putative chitin synthase (CHS) sequences are decreasingly present in Crustacea (39%), Stramenopiles (16%) and Insecta (14%) from the Marine Atlas of Tara Oceans Unigenes version 1 Metatranscriptomes (MATOUv1+T) database. A CHS gene from the model diatom Thalassiosira pseudonana (Thaps3_J4413, designated TpCHS1) was identified. Homology analysis of TpCHS1 in Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP), PhycoCosm, and the PLAZA diatom omics data set showed that Mediophyceae and Thalassionemales species were potential new ß-chitin producers besides Thalassiosirales. TpCHS1 was overexpressed in Saccharomyces cerevisiae and Phaeodactylum tricornutum. In transgenic P. tricornutum lines, TpCHS1-eGFP localizes to the Golgi apparatus and plasma membrane and predominantly accumulates in the cleavage furrow during cell division. Enhanced TpCHS1 expression could induce abnormal cell morphology and reduce growth rates in P. tricornutum, which might be ascribed to the inhibition of the G2/M phase. S. cerevisiae was proved to be a better system for expressing large amounts of active TpCHS1, which effectively incorporates UDP-N-acetylglucosamine in radiometric in vitro assays. Our study expands the knowledge on chitin synthase taxonomic distribution in marine eukaryotic microbes, and is the first to collectively characterize an active marine diatom CHS which may play an important role during cell division. IMPORTANCE As the most abundant biopolymer in the oceans, the significance of chitin and its biosynthesis is rarely demonstrated in diatoms, which are the main contributors to the primary productivity of the oceans, ascribed to their huge biomass and efficient photosynthesis. We retrieved genes involved in chitin-based metabolism against the Tara Oceans Gene Atlas to expand our knowledge about their diversity and distribution in the marine environment. Potential new producers of chitin were found from the analysis of various algal transcriptome and genome databases. Heterologous expression confirms that Thalassiosira pseudonana contains an active chitin synthase (CHS) which may play an important role in the cell division process of diatoms. This study provides new insight into CHS geographic and taxonomic distribution in marine eukaryotic microbes, as well as into a new CHS functioning in the biosynthesis of ß-chitin in diatoms.

The Cell Wall Polysaccharides Biosynthesis in Seaweeds: A Molecular Perspective.

Cell wall polysaccharides (CWPS) of seaweeds play crucial roles in mechanical shear resistance, cell-cell adhesion and the interactions with changeable marine environments. They have diverse applications in food, cosmetics, agriculture, pharmaceuticals and therapeutics. The recent boost of multi-omics sequence analysis has rapidly progressed the mining of presumed genes encoding enzymes involved in CWPS biosynthesis pathways. In this review, we summarize the biosynthetic pathways of alginate, fucoidan, agar, carrageenan and ulvan in seaweeds referred to the literatures on published genomes and biochemical characterization of encoded enzymes. Some transcriptomic data were briefly reported to discuss the correlation between gene expression levels and CWPS contents. Mannuronan C-5 epimerase (MC5E) and carbohydrate sulfotransferase (CST) are crucial enzymes for alginate and sulfated CWPS, respectively. Nonetheless, most CWPS-relevant genes were merely investigated by gene mining and phylogenetic analysis. We offer an integrative view of CWPS biosynthesis from a molecular perspective and discuss about the underlying regulation mechanism. However, a clear understanding of the relationship between chemical structure and bioactivities of CWPS is limited, and reverse genetic manipulation and effective gene editing tools need to be developed in future.

Glutamate Dehydrogenase Functions in Glutamic Acid Metabolism and Stress Resistance in Pyropia haitanensis.

Pyropia haitanensis is an important laver species in China. Its quality traits are closely related to the content of glutamic acid. Glutamate dehydrogenase (GDH) is a crucial enzyme in the glutamic acid metabolism. In this study, two GDH genes from P. haitanensis, PhGDH1 and PhGDH2, were cloned and successfully expressed in Escherichia coli. The in vitro enzyme activity assay demonstrated that the catalytic activity of PhGDHs is mainly in the direction of ammonium assimilation. The measured Km values of PhGDH1 for NADH, (NH4)2SO4, and α-oxoglutarate were 0.12, 4.99, and 0.16 mM, respectively, while the corresponding Km values of PhGDH2 were 0.02, 3.98, and 0.104 mM, respectively. Site-directed mutagenesis results showed that Gly193 and Thr361 were important catalytic residues for PhGDH2. Moreover, expression levels of both PhGDHs were significantly increased under abiotic stresses. These results suggest that PhGDHs can convert α-oxoglutarate to glutamic acid, and enhance the flavor and stress resistance of P. haitanensis.

Full-Length Transcriptome of Thalassiosira weissflogii as a Reference Resource and Mining of Chitin-Related Genes.

ß-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of ß-chitin-derived high-value enzymes and products.

Genome-wide identification of chitinase genes in Thalassiosira pseudonana and analysis of their expression under abiotic stresses.

BACKGROUND: The nitrogen-containing polysaccharide chitin is the second most abundant biopolymer on earth and is found in the cell walls of diatoms, where it serves as a scaffold for biosilica deposition. Diatom chitin is an important source of carbon and nitrogen in the marine environment, but surprisingly little is known about basic chitinase metabolism in diatoms. RESULTS: Here, we identify and fully characterize 24 chitinase genes from the model centric diatom Thalassiosira pseudonana. We demonstrate that their expression is broadly upregulated under abiotic stresses, despite the fact that chitinase activity itself remains unchanged, and we discuss several explanations for this result. We also examine the potential transcriptional complexity of the intron-rich T. pseudonana chitinase genes and provide evidence for two separate tandem duplication events during their evolution. CONCLUSIONS: Given the many applications of chitin and chitin derivatives in suture production, wound healing, drug delivery, and other processes, new insight into diatom chitin metabolism has both theoretical and practical value.

Genome-wide analysis of the Saccharina japonica sulfotransferase genes and their transcriptional profiles during whole developmental periods and under abiotic stresses.

BACKGROUND: As a unique sulfated polysaccharide, fucoidan is an important component of cell wall in brown seaweeds. Its biochemical properties are determined by the positions and quantity of sulfate groups. Sulfotransferases (STs) catalyze the sulfation process, which transfer the sulfuryl groups to carbohydrate backbones and are crucial for fucoidan biosynthesis. Nevertheless, the structures and functions of STs in brown seaweeds are rarely investigated. RESULTS: There are a total of 44 ST genes identified from our genome and transcriptome analysis of Saccharina japonica, which were located in the 17 scaffolds and 11 contigs. The S. japonica ST genes have abundant introns and alternative splicing sites, and five tandem duplicated gene clusters were identified. Generally, the ST genes could be classified into five groups (Group I ~ V) based on phylogenetic analysis. Accordingly, the ST proteins, which were encoded by genes within the same group, contained similar conserved motifs. Members of the S. japonica ST gene family show various expression patterns in different tissues and developmental stages. Transcriptional profiles indicate that the transcriptional levels of more than half of the ST genes are higher in kelp basal blades than in distal blades. Except for ST5 and ST28, most ST genes are down-regulated with the kelp development stages. The expression levels of nine ST genes were detected by real-time quantitative PCR, which demonstrates that they responded to low salinity and drought stresses. CONCLUSIONS: Various characteristics of the STs allow the feasibilities of S. japonica to synthesize fucoidans with different sulfate groups. This enables the kelp the potential to adapt to the costal environments and meet the needs of S. japonica growth.

Transcriptome sequencing of Saccharina japonica sporophytes during whole developmental periods reveals regulatory networks underlying alginate and mannitol biosynthesis.

BACKGROUND: Alginate is an important cell wall component and mannitol is a soluble storage carbon substance in the brown seaweed Saccharina japonica. Their contents vary with kelp developmental periods and harvesting time. Alginate and mannitol regulatory networks and molecular mechanisms are largely unknown. RESULTS: With WGCNA and trend analysis of 20,940 known genes and 4264 new genes produced from transcriptome sequencing of 30 kelp samples from different stages and tissues, we deduced that ribosomal proteins, light harvesting complex proteins and "imm upregulated 3" gene family are closely associated with the meristematic growth and kelp maturity. Moreover, 134 and 6 genes directly involved in the alginate and mannitol metabolism were identified, respectively. Mannose-6-phosphate isomerase (MPI2), phosphomannomutase (PMM1), GDP-mannose 6-dehydrogenase (GMD3) and mannuronate C5-epimerase (MC5E70 and MC5E122) are closely related with the high content of alginate in the distal blade. Mannitol accumulation in the basal blade might be ascribed to high expression of mannitol-1-phosphate dehydrogenase (M1PDH1) and mannitol-1-phosphatase (M1Pase) (in biosynthesis direction) and low expression of mannitol-2-dehydrogenase (M2DH) and Fructokinase (FK) (in degradation direction). Oxidative phosphorylation and photosynthesis provide ATP and NADH for mannitol metabolism whereas glycosylated cycle and tricarboxylic acid (TCA) cycle produce GTP for alginate biosynthesis. RNA/protein synthesis and transportation might affect alginate complex polymerization and secretion processes. Cryptochrome (CRY-DASH), xanthophyll cycle, photosynthesis and carbon fixation influence the production of intermediate metabolite of fructose-6-phosphate, contributing to high content of mannitol in the basal blade. CONCLUSIONS: The network of co-responsive DNA synthesis, repair and proteolysis are presumed to be involved in alginate polymerization and secretion, while upstream light-responsive reactions are important for mannitol accumulation in meristem of kelp. Our transcriptome analysis provides new insights into the transcriptional regulatory networks underlying the biosynthesis of alginate and mannitol during S. japonica developments.

Verification of the Saccharina japonica Translocon Tic20 and its Localization in the Chloroplast Membrane in Diatoms.

Tic20 is an important translocon protein that plays a role in protein transport in the chloroplast. The sequence of Tic20 was determined in the lower brown alga Saccharina japonica. Structural analysis of SjTic20 revealed a noncanonical structure consisting of an N-terminal non-cyanobacterium-originated EF-hand domain (a helix-loop-helix structural domain) and a C-terminal cyanobacterium-originated Tic20 domain. Subcellular localization and transmembrane analysis indicated that SjTic20 featured an "M"-type Nin-Cin-terminal orientation, with four transmembrane domains in the innermost membrane of the chloroplast in the microalga Phaeodactylum tricornutum, and the EF-hand domain was entirely extruded into the chloroplast stroma. Our study provides information on the structure, localization, and topological features of SjTic20, and further functional analysis of SjTic20 in S. japonica is needed.

Genome-wide identification of genes involved in carbon fixation in Saccharina japonica and responses of putative C4-related genes to bicarbonate concentration and light intensity.

Brown algae play a dominant role in the primary productivity of coastal ecosystems and may have an efficient carbon fixation. In this work, 56 genes involved in inorganic carbon fixation were identified from the Saccharina japonica genome. Sequence structure analysis of these genes showed the existence of corresponding function domains and active amino acid sites highly conserved with other stramenopile species. The predicted subcellular localizations showed that Calvin cycle-related enzymes predominantly reside in the plastid and that putative C4-related enzymes are mainly distributed in the mitochondrion. We determined the transcriptional profiles and enzymatic activities of these C4-related enzymes in response to the KHCO3 concentrations and light intensities. Pyruvate orthophosphate dikinase (PPDK) presented the greatest response to low HCO3- concentrations and high light intensity. Phosphoenolpyruvate carboxykinase (PEPCK) was up-regulated at low HCO3- concentrations to compensate for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and might be the crucial decarboxylase in this kelp. We propose that S. japonica might possess a PPDK- and PEPCK-dependent C4-like pathway that enables its rapid growth in natural coastal environments.

Comparative characterization of putative chitin deacetylases from Phaeodactylum tricornutum and Thalassiosira pseudonana highlights the potential for distinct chitin-based metabolic processes in diatoms.

Chitin is generally considered to be present in centric diatoms but not in pennate species. Many aspects of chitin biosynthetic pathways have not been explored in diatoms. We retrieved chitin metabolic genes from pennate (Phaeodactylum tricornutum) and centric (Thalassiosira pseudonana) diatom genomes. Chitin deacetylase (CDA) genes from each genome (PtCDA and TpCDA) were overexpressed in P. tricornutum. We performed comparative analysis of their sequence structure, phylogeny, transcriptional profiles, localization and enzymatic activities. The chitin relevant proteins show complex subcellular compartmentation. PtCDA was likely acquired by horizontal gene transfer from prokaryotes, whereas TpCDA has closer relationships with sequences in Opisthokonta. Using transgenic P. tricornutum lines expressing CDA-green fluorescent protein (GFP) fusion proteins, PtCDA predominantly localizes to Golgi apparatus whereas TpCDA localizes to endoplasmic reticulum/chloroplast endoplasmic reticulum membrane. CDA-GFP overexpression upregulated the transcription of chitin synthases and potentially enhanced the ability of chitin synthesis. Although both CDAs are active on GlcNAc5 , TpCDA is more active on the highly acetylated chitin polymer DA60. We have addressed the ambiguous characters of CDAs from P. tricornutum and T. pseudonana. Differences in localization, evolution, expression and activities provide explanations underlying the greater potential of centric diatoms for chitin biosynthesis. This study paves the way for in vitro applications of novel CDAs.

Identification and characterization of Prunus persica miRNAs in response to UVB radiation in greenhouse through high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression of target mRNAs involved in plant growth, development, and abiotic stress. As one of the most important model plants, peach (Prunus persica) has high agricultural significance and nutritional values. It is well adapted to be cultivated in greenhouse in which some auxiliary conditions like temperature, humidity, and UVB etc. are needed to ensure the fruit quality. However, little is known about the genomic information of P. persica under UVB supplement. Transcriptome and expression profiling data for this species are therefore important resources to better understand the biological mechanism of seed development, formation and plant adaptation to environmental change. Using a high-throughput miRNA sequencing, followed by qRT-PCR tests and physiological properties determination, we identified the responsive-miRNAs under low-dose UVB treatment and described the expression pattern and putative function of related miRNAs and target genes in chlorophyll and carbohydrate metabolism. RESULTS: A total of 164 known peach miRNAs belonging to 59 miRNA families and 109 putative novel miRNAs were identified. Some of these miRNAs were highly conserved in at least four other plant species. In total, 1794 and 1983 target genes for known and novel miRNAs were predicted, respectively. The differential expression profiles of miRNAs between the control and UVB-supplement group showed that UVB-responsive miRNAs were mainly involved in carbohydrate metabolism and signal transduction. UVB supplement stimulated peach to synthesize more chlorophyll and sugars, which was verified by qRT-PCR tests of related target genes and metabolites' content measurement. CONCLUSION: The high-throughput sequencing data provided the most comprehensive miRNAs resource available for peach study. Our results identified a series of differentially expressed miRNAs/target genes that were predicted to be low-dose UVB-responsive. The correlation between transcriptional profiles and metabolites contents in UVB supplement groups gave novel clues for the regulatory mechanism of miRNAs in Prunus. Low-dose UVB supplement could increase the chlorophyll and sugar (sorbitol) contents via miRNA-target genes and therefore improve the fruit quality in protected cultivation of peaches.

Comparative characterization of two GDP-mannose dehydrogenase genes from Saccharina japonica (Laminariales, Phaeophyceae).

BACKGROUND: Saccharina japonica is an important commercial brown seaweed, its main product is alginate, which is used in food, textile and by the cosmetic and pharmaceutical industries. GDP-mannose dehydrogenase (GMD) is the key enzyme involved in the synthesis of alginate. However, little is known about GMD in S. japonica. Here we report comparative biochemical analysis of two GMD genes in S. japonica. RESULTS: Two GMD genes from S. japonica (Sjgmd1, Sjgmd2) were cloned. The open reading frame lengths of Sjgmd1, Sjgmd2 are 963 bp and 948 bp, respectively. Alignment analysis showed that the two SjGMD sequences shared 79.38 % identity. Both proteins possess the GGxCLPKDV and GxGxVG sequence motifs characteristic of the short-chain dehydrogenase/reductase superfamily. The optimum temperatures for SjGMDs were 30 °C (SjGMD1) and 20 °C (SjGMD2), and the optimum pH values were 8.0 (SjGMD1) and 8.25 (SjGMD2). Kinetic analysis demonstrated the Km values for the substrate GDP-mannose were 289 µM (SjGMD1) and 177 µM (SjGMD2), and the Km values for the cofactor NAD(+) were 139 µM (SjGMD1) and 195 µM (SjGMD2). The metal iron Zn(2+) is a potent inhibitor of SjGMD1 and SjGMD2. Real-time PCR analysis showed that heat and desiccation treatments resulted in a significant increase in Sjgmd1 and Sjgmd2 transcript abundance, suggesting that the SjGMDs are directly involved in the acclimitisation of S. japonica to abiotic stresses. CONCLUSION: Our work identified two novel genes encoding GMD in S. japonica, comparatively characterized their structural characteristics and enzyme kinetics, and revealed the function of GMD in the stress adaptability of S. japonica. The knowledge obtained here enriched our understanding of the alginate synthesis mechanism in S. japonica, and may promote further research on functional differences between GMD genes.

Characterization of mannitol-2-dehydrogenase in Saccharina japonica: evidence for a new polyol-specific long-chain dehydrogenases/reductase.

Mannitol plays a crucial role in brown algae, acting as carbon storage, organic osmolytes and antioxidant. Transcriptomic analysis of Saccharina japonica revealed that the relative genes involved in the mannitol cycle are existent. Full-length sequence of mannitol-2-dehydrogenase (M2DH) gene was obtained, with one open reading frame of 2,007 bp which encodes 668 amino acids. Cis-regulatory elements for response to methyl jasmonic acid, light and drought existed in the 5'-upstream region. Phylogenetic analysis indicated that SjM2DH has an ancient prokaryotic origin, and is probably acquired by horizontal gene transfer event. Multiple alignment and spatial structure prediction displayed a series of conserved functional residues, motifs and domains, which favored that SjM2DH belongs to the polyol-specific long-chain dehydrogenases/reductase (PSLDR) family. Expressional profiles of SjM2DH in the juvenile sporophytes showed that it was influenced by saline, oxidative and desiccative factors. SjM2DH was over-expressed in Escherichia coli, and the cell-free extracts with recombinant SjM2DH displayed high activity on D-fructose reduction reaction. The analysis on SjM2DH gene structure and biochemical parameters reached a consensus that activity of SjM2DH is NADH-dependent and metal ion-independent. The characterization of SjM2DH showed that M2DH is a new member of PSLDR family and play an important role in mannitol metabolism in S. japonica.

Mannitol metabolism in brown algae involves a new phosphatase family.

Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase and a mannitol-1-phosphatase (M1Pase). Genome analysis of the brown algal model Ectocarpus siliculosus allowed identification of genes potentially involved in the mannitol cycle. Among these, two genes coding for haloacid dehalogenase (HAD)-like enzymes were suggested to correspond to M1Pase activity, and thus were named EsM1Pase1 and EsM1Pase2, respectively. To test this hypothesis, both genes were expressed in Escherichia coli. Recombinant EsM1Pase2 was shown to hydrolyse the phosphate group from mannitol-1-phosphate to produce mannitol but was not active on the hexose monophosphates tested. Gene expression analysis showed that transcription of both E. siliculosus genes was under the influence of the diurnal cycle. Sequence analysis and three-dimensional homology modelling indicated that EsM1Pases, and their orthologues in Prasinophytes, should be seen as founding members of a new family of phosphatase with original substrate specificity within the HAD superfamily of proteins. This is the first report describing the characterization of a gene encoding M1Pase activity in photosynthetic organisms.

Chloroplast genome of one brown seaweed, Saccharina japonica (Laminariales, Phaeophyta): its structural features and phylogenetic analyses with other photosynthetic plastids.

The chloroplast genome sequence of one brown seaweed, Saccharina japonica, was fully determined. It is characterized by 130,584 base pairs (bp) with a large and a small single-copy region (LSC and SSC), separated by two copies of inverted repeats (IR1 and IR2). The inverted repeat is 5015 bp long, and the sizes of SSC and LSC are 43,174 bp and 77,378 bp, respectively. The chloroplast genome of S. japonica consists of 139 protein-coding genes, 29 tRNA genes, and 3 ribosomal RNA genes. One intron was found in one tRNA-Leu gene in the chloroplast genome of S. japonica. Four types of overlapping genes were identified, ycf24 overlapped with ycf16 by 4 nucleotides (nt), ftrB overlapped with ycf12 by 6 nt, rpl4 and rpl23 overlapped by 8 nt, finally, psbC overlapped with psbD by 53 nt. With two sets of concatenated plastid protein data, 40-protein dataset and 26-protein dataset, the chloroplast phylogenetic relationship among S. japonica and the other photosynthetic species was evaluated. We found that the chloroplast genomes of haptophyte, cryptophyte and heterokont were not resolved into one cluster by the 40-protein dataset with amino acid composition bias, although it was recovered with strong support by the 26-protein dataset.

QTL mapping for frond length and width in Laminaria japonica aresch (Laminarales, Phaeophyta) using AFLP and SSR markers.

In Laminaria japonica Aresch breeding practice, two quantitative traits, frond length (FL) and frond width (FW), are the most important phenotypic selection index. In order to increase the breeding efficiency by integrating phenotypic selection and marker-assisted selection, the first set of QTL controlling the two traits were determined in F(2) family using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Two prominent L. japonicas inbred lines, one with "broad and thin blade" characteristics and another with "long and narrow blade" characteristics, were applied in the hybridization to yield the F(2) mapping population with 92 individuals. A total of 287 AFLP markers and 11 SSR markers were used to construct a L. japonica genetic map. The yielded map was consisted of 28 linkage groups (LG) named LG1 to LG28, spanning 1,811.1 cM with an average interval of 6.7 cM and covering the 82.8% of the estimated genome 2,186.7 cM. While three genome-wide significant QTL were detected on LG1 (two QTL) and LG4 for "FL," explaining in total 42.36% of the phenotypic variance, two QTL were identified on LG3 and LG5 for the trait "FW," accounting for the total of 36.39% of the phenotypic variance. The gene action of these QTL was additive and partially dominant. The yielded linkage map and the detected QTL can provide a tool for further genetic analysis of two traits and be potential for maker-assisted selection in L. japonica breeding.