The O-linked-N -acetylglucosamine (O-GlcNAc) modifi cation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the aetiology of diabetes and neurodegeneration. This intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, OGT. Here we report a novel OGT, EOGT, responsible for extracellular O-GlcNAcylation. Although both OGT and EOGT are regulated by hexosamine fl ux, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. Loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy (Dp), a membrane-anchored extracellular protein, is O-GlcNAcylated, and EOGT is required for Dp-dependent epithelial cell -matrix interactions. Thus, O-GlcNAcylation of secreted and membrane glycoproteins is a novel mediator of cell -cell or cell -matrix interactions at the cell surface.

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N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca -regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5-CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5-CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca signaling processes, and demonstrate their requirement for guard cell anion channel regulation.

The Pacific bluefin tuna, Thunnus orientalis, is a highly migratory species that is widely distributed in the North Pacific Ocean. Like other marine species, T. orientalis has no external sexual dimorphism; thus, identifying sex-specific variants from whole genome sequence data is a useful approach to develop an effective sex identification method. Here, we report an improved draft genome of T. orientalis and male-specific DNA markers. Combining PacBio long reads and Illumina short reads sufficiently improved genome assembly, with a 38-fold increase in scaffold contiguity (to 444 scaffolds) compared to the first published draft genome. Through analysing re-sequence data of 15 males and 16 females, 250 male-specific SNPs were identified from more than 30 million polymorphisms. All male-specific variants were male-heterozygous, suggesting that T. orientalis has a male heterogametic sex-determination system. The largest linkage disequilibrium block (3,174 bp on scaffold_064) contained 51 male-specific variants. PCR primers and a PCR-based sex identification assay were developed using these male-specific variants. The sex of 115 individuals (56 males and 59 females; sex was diagnosed by visual examination of the gonads) was identified with high accuracy using the assay. This easy, accurate, and practical technique facilitates the control of sex ratios in tuna farms. Furthermore, this method could be used to estimate the sex ratio and/or the sex-specific growth rate of natural populations.

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. Well-known examples of such modification are O-linked fucose (O-fucose) and O-linked glucose (O-glucose) glycans on epidermal growth factor (EGF) domains. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as urinary-type plasminogen activator and Notch receptors. Two glycosyltransferases catalyze the initiation and elongation of O-fucose glycans. The initiation process is catalyzed by O-fucosyltransferase 1, which is essential for Notch signalling in both Drosophila and mice. O-fucosyltransferase 1 can affect the folding, ligand interaction and endocytosis of Notch receptors, and both the glycosyltransferase and non-catalytic activities of O-fucosyltransferase 1 have been reported. The elongation of O-fucose monosaccharide is catalyzed by Fringe-related genes, which differentially modulate the interaction between Notch and two classes of ligands, namely, Delta and Serrate/Jagged. In this article, we have reviewed the recent reports addressing the distinctive features of the glycosyltransferases and O-glycans present on the EGF domains.

Seriola quinqueradiata (yellowtail) is the most widely farmed and economically important fish in aquaculture in Japan. In this study, we used the genome of haploid yellowtail fish larvae for de novo assembly of whole-genome sequences, and built a high-quality draft genome for the yellowtail. The total length of the assembled sequences was 627.3 Mb, consisting of 1,394 scaffold sequences (>2 kb) with an N50 length of 1.43 Mb. A total of 27,693 protein-coding genes were predicted for the draft genome, and among these, 25,832 predicted genes (93.3%) were functionally annotated. Given our lack of knowledge of the yellowtail digestive system, and using the annotated draft genome as a reference, we conducted an RNA-Seq analysis of its three digestive organs (stomach, intestine and rectum). The RNA-Seq results highlighted the importance of certain genes in encoding proteolytic enzymes necessary for digestion and absorption in the yellowtail gastrointestinal tract, and this finding will accelerate development of formulated feeds for this species. Since this study offers comprehensive annotation of predicted protein-coding genes, it has potential broad application to our understanding of yellowtail biology and aquaculture.

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.

The Japanese eel (Anguilla japonica) is among the most important aquaculture fish species in Eastern Asia. The present study aimed to identify the genetic parameters underlying body size and the timing at metamorphosis from leptocephali to glass eels in captive-bred Japanese eels, with the intent to foster sustainable development. Larvae from a partly factorial cross (14 sires × 11 dams) were reared until the point of metamorphosis into glass eels. In these organisms, we observed moderate heritability and mild genetic correlations among traits related to body size (h2 = 0.16–0.33) and timing at metamorphosis (h2 = 0.36–0.41). In an F1 full-sib family, quantitative trait loci (QTL) mapping for these traits identified one significant (genome-wide P < 0.05) and five suggestive QTLs (chromosome-wide P < 0.05). These results suggest that in the Japanese eel, metamorphic traits exhibit a polygenic genetic structure comprising many QTLs with small effects. In addition, we updated the genetic linkage map for the Japanese eel and integrated it with our newly constructed de novo genome assembly. The information and tools generated from this study will contribute to the development of freshwater eel genetics and genomics.