Abstract Background As a unique sulfated polysaccharide, fucoidan is the major component of cell wall in brown seaweeds. Its biochemical properties are determined by the positions and quantity of sulfate groups. The sulfation process is catalyzed by sulfotransferases (STs), 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 not completely understood.Results There are a total of 27 ST genes identified from our genome and transcriptome analysis of Saccharina japonica and they were located in the 12 scaffolds and seven contigs. The S. japonica ST genes have many introns and alternative splicing sites, and four tandem duplicated gene clusters were identified among this genes family. Generally, the ST genes could be classified into five groups (Group I~V) based on phylogenetic analysis, and the ST proteins, which were encoded by genes within the same group, contained similar conserved motifs. In group I sequences, we found two highly conserved regions (region I: TxPKSGTxW; region IV: RKGxxGDWKxxFT), and Lys 81 and Arg 287 are critical for catalysis and substrate binding. Members of the S. japonica ST gene family show various expression patterns in different tissues and developmental stages. Transcripttional profiles indicate that more than half of ST genes transcriptional levels in kelp basal blades are higher than in distal blades, and decrease with the kelp development stages, while only ST 5 , 9 , 12 , 18 and 25 are increased. The co-down-regulated pathways are related to oxidative phosphorylation, fatty acid biosynthesis and metabolic pathways, while the up-regulated pathways are involved with ribosome, nitrogen and sulfur metabolism.Conclusion Various characteristics of the STs allow the feasibilities of S. japonica to adapt to the costal environments, meet the needs of S. japonica growth and support their giant blades. This also reflects the complexity of fucoidan biosynthesis in different marine environments, tissues, and developmental stages.
Abstract 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.
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.
Salinity is a serious threat to most land plants. Although seaweeds adapt to salty environments, intertidal species experience wide fluctuations in external salinities, including hyper- and hypo-saline stress. Bangia fuscopurpurea is an economic intertidal seaweed with a strong tolerance to hypo-salinity. Until now, the salt stress tolerance mechanism has remained elusive. Our previous study showed that the expression of B. fuscopurpurea plasma membrane H+-ATPase (BfPMHA) genes were the most upregulated under hypo-salinity. In this study, we obtained the complete sequence of BfPMHA, traced the relative expression of this BfPMHA gene in B. fuscopurpurea under hypo-salinity, and analyzed the protein structure and properties based on the gene's sequence. The result showed that the expression of BfPMHA in B. fuscopurpurea increased significantly with varying hypo-salinity treatments, and the higher the degree of low salinity stress, the higher the expression level. This BfPMHA had typical PMHA structures with a Cation-N domain, an E1-E2 ATPase domain, a Hydrolase domain, and seven transmembrane domains. In addition, through the membrane system yeast two-hybrid library, three candidate proteins interacting with BfPMHA during hypo-saline stress were screened, fructose-bisphosphate aldolase (BfFBA), glyceraldehyde 3-phosphate dehydrogenase (NADP+) (phosphorylating) (BfGAPDH), and manganese superoxide dismutase (BfMnSOD). The three candidates and BfPMHA genes were successfully transferred and overexpressed in a BY4741 yeast strain. All of them significantly enhanced the yeast tolerance to NaCl stress, verifying the function of BfPMHA in salt stress response. This is the first study to report the structure and topological features of PMHA in B. fuscopurpurea and its candidate interaction proteins in response to salt stress.
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