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The article examines the biosynthesis of 2-phenylethanol, the main floral scent compound in roses, using assays of a proteolipid apoprotein (PLP)-dependent decarboxylase and a phenylacetaldehyde reductase (PAR). According to the authors, the activity of rose petal L-aromatic amino acid decarboxylase (AADC) to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer. They reveal that phenylacetaldehyde was converted to 2-phenylethanol by the action of PAR.

1,3,5-Trimethoxybenzene is a key component of the Chinese rose odor. This compound is synthesized in three successive methylation steps from phloroglucinol, the initial precursor. A novel, to our knowledge, phloroglucinol O-methyltransferase (POMT) characterized here methylates the first step to produce the intermediate 3,5-dihydroxyanisole, while two previously described orcinol O-methyltransferases catalyze the subsequent steps. We isolated POMT from rose petals and determined partial amino acid sequences of the purified enzyme. The full-length POMT cDNA was isolated and expressed in Escherichia coli. Both the native and recombinant POMT exhibited substrate specificity for phloroglucinol. POMT was expressed specifically in floral organs, in accordance with its role as a key enzyme in the synthesis of rose floral scent compounds. [ABSTRACT FROM AUTHOR]

Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn(II)-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe(II). Affinity and catalytic constants were estimated at 470 μM and 4.69 sec−1, respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe(II), not Zn(II), is essential for POD activity and that Mn(II) could partially fulfill the function of Fe(II). A mutant POD with replacement of His183, corresponding to one of three Zn(II)-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe(II) as a catalytic center instead of Zn(II). A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe(II)-dependent dioxygenase. [ABSTRACT FROM AUTHOR]

Background:Pleurocybella porrigens is a mushroom-forming fungus, which has been consumed as a traditional food in Japan. In 2004, 55 people were poisoned by eating the mushroom and 17 people among them died of acute encephalopathy. Since then, the Japanese government has been alerting Japanese people to take precautions against eating the P. porrigens mushroom. Unfortunately, despite efforts, the molecular mechanism of the encephalopathy remains elusive. The genome and transcriptome sequence data of P. porrigens and the related species, however, are not stored in the public database. To gain the omics data in P. porrigens, we sequenced genome and transcriptome of its fruiting bodies and mycelia by next generation sequencing. Methodology/Principal Findings:Short read sequences of genomic DNAs and mRNAs in P. porrigens were generated by Illumina Genome Analyzer. Genome short reads were de novo assembled into scaffolds using Velvet. Comparisons of genome signatures among Agaricales showed that P. porrigens has a unique genome signature. Transcriptome sequences were assembled into contigs (unigenes). Biological functions of unigenes were predicted by Gene Ontology and KEGG pathway analyses. The majority of unigenes would be novel genes without significant counterparts in the public omics databases. Conclusions:Functional analyses of unigenes present the existence of numerous novel genes in the basidiomycetes division. The results mean that the omics information such as genome, transcriptome and metabolome in basidiomycetes is short in the current databases. The large-scale omics information on P. porrigens, provided from this research, will give a new data resource for gene discovery in basidiomycetes. [ABSTRACT FROM AUTHOR]

Biohydrogen is mainly produced by anaerobic bacteria, anaerobic fungi, and algae under anaerobic conditions. In higher eukaryotes, it is thought that molecular hydrogen (H 2 ) functions as a signaling molecule for physiological processes such as stress responses. Here, it is demonstrated that white-rot fungi produce H 2 during wood decay. The white-rot fungus Trametes versicolor produces H 2 from wood under aerobic conditions, and H 2 production is completely suppressed under hypoxic conditions. Additionally, oxalate and formate supplementation of the wood culture increased the level of H 2 evolution. RNA-seq analyses revealed that T. versicolor oxalate production from the TCA/glyoxylate cycle was down-regulated, and conversely, genes encoding oxalate and formate metabolism enzymes were up-regulated. Although the involvement in H 2 production of a gene annotated as an iron hydrogenase was uncertain, the results of organic acid supplementation, gene expression, and self-recombination experiments strongly suggest that formate metabolism plays a role in the mechanism of H 2 production by this fungus. It is expected that this novel finding of aerobic H 2 production from wood biomass by a white-rot fungus will open new fields in biohydrogen research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mori, Takahashi, Soga, Arimoto, Kishikawa, Yama, Dohra, Kawagishi and Hirai.)

A novel bacterium , designated 5-21a T , isolated from chitin-treated upland soil, exhibits methionine (Met) auxotrophy and chitinolytic activity. A physiological experiment revealed the cobalamin (synonym, vitamin B 12 )(Cbl)-auxotrophic property of strain 5-21a T . The newly determined complete genomic sequence indicated that strain 5-21a T possesses only the putative gene for Cbl-dependent Met synthase (MetH) and lacks that for the Cbl-independent one (MetE), which implies the requirement of Cbl for Met-synthesis in strain 5-21a T . The set of genes for the upstream (corrin ring synthesis) pathway of Cbl synthesis is absent in the genome of strain 5-21a T , which explains the Cbl-auxotrophy of 5-21a T . This strain was characterized via a polyphasic approach to determine its taxonomic position. The nucleotide sequences of two copies of the 16S rRNA gene of strain 5-21a T indicated the highest similarities to Lysobacter soli DCY21 T (99.8 and 99.9 %) and Lysobacter panacisoli CJ29 T (98.7 and 98.8 %, respectively), whose Cbl-auxotrophic properties were revealed in this study. The principal respiratory quinone was Q-8. The predominant cellular fatty acids were iso-C 15:0 , iso-C 16:0 and iso-C 17:1 ω 9 c . The complete genome sequence of strain 5-21a T revealed that the genome size was 4 155 451 bp long and the G+C content was 67.87 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain 5-21a T and its most closely phylogenetic relative L. soli DCY21 T were 88.8 and 36.5%, respectively. Based on genomic, chemotaxonomic, phenotypic and phylogenetic data, strain 5-21a T represents a novel species in the genus Lysobacter , for which the name Lyobacter auxotrophicus sp. nov. is proposed. The type strain is 5-21a T (=NBRC 115507 T =LMG 32660 T ).

We report here 16S rRNA gene amplicon sequence analysis of the gut microbiota in three species of deep-sea fish collected from Suruga Bay, Japan. Of the three species, two were dominated by the phylum Proteobacteria (genus Photobacterium ), while one was dominated by the phyla Spirochaetes (genus Brevinema ) and Tenericutes (unclassified Mycoplasmataceae )., (Copyright © 2021 Iwatsuki et al.)

The collapse of Comamonas testosteroni R2 under chemostat conditions and the aerobic growth of strain R2 under batch conditions with phenol as the sole carbon source were investigated using physiological and transcriptomic techniques. Phenol-/catechol-degrading activities under chemostat conditions gradually decreased, suggesting that metabolites produced from strain R2 accumulated in the culture, which caused negative feedback. The competitive inhibition of phenol hydroxylase and catechol dioxygenase was observed in a crude extract of the supernatant collected from the collapsed culture. Transcriptomic analyses showed that genes related to nitrogen transport were up-regulated; the ammonium transporter amtB was up-regulated approximately 190-fold in the collapsed status, suggesting an increase in the concentration of ammonium in cells. The transcriptional levels of most of the genes related to gluconeogenesis, glycolysis, the pentose phosphate pathway, and the TCA and urea cycles decreased by ~0.7-fold in the stable status, whereas the activities of glutamate synthase and glutamine synthetase increased by ~2-fold. These results suggest that ammonium was assimilated into glutamate and glutamine via 2-oxoglutarate under the limited supply of carbon skeletons, whereas the synthesis of other amino acids and nucleotides was repressed by 0.6-fold. Furthermore, negative feedback appeared to cause an imbalance between carbon and nitrogen metabolism, resulting in collapse. The effects of amino acids on negative feedback were investigated. L-arginine allowed strain R2 to grow normally, even under growth-inhibiting conditions, suggesting that the imbalance was corrected by the stimulation of the urea cycle, resulting in the rescue of strain R2.

Linear azole-containing peptides are a class of ribosomally synthesized and post-translationally modified peptides. We performed a chemical investigation on marine actinomycetes, and new linear azole-containing peptides named spongiicolazolicins A and B were found in the MeOH extracts of a newly isolated strain Streptomyces sp. CWH03 (NBRC 114659) and two strains of S. spongiicola (strain HNM0071 T : DSM 103383 T and strain 531S: NBRC 113560). The strain Streptomyces sp. CWH03 was indicated to be a new species closely related to S. spongiicola by phylogenetic analysis using the genome sequence. The new peptides named spongiicolazolicins A and B were isolated from the cell of Streptomyces sp. CWH03. The partial structure of spongiicolazolicin A was determined by 2D NMR experiments. Based on data of MS/MS experiments, the chemical structures of spongiicolazolicins A and B were proposed using the amino acid sequence deduced from the precursor-encoding gene, which was found from whole-genome sequence data of Streptomyces sp. CWH03. The biosynthetic gene cluster of spongiicolazolicins was proposed based on comparative analysis with that of a known linear azole peptide goadsporin. KEY POINTS: • Streptomyces sp. CWH03 was a new species isolated from marine sediment. • New linear azole-containing peptides named spongiicolazolicins A and B were isolated. • Biosynthetic pathway of spongiicolazolicins was proposed.

Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-D-glucose by LgdB2; next, LgdC reduces 3-keto-D-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

Deleted in lung and esophageal cancer 1 (DLEC1) is a tumour suppressor gene that is downregulated in various cancers in humans; however, the physiological and molecular functions of DLEC1 are still unclear. This study investigated the critical role of Dlec1 in spermatogenesis and male fertility in mice. Dlec1 was significantly expressed in testes, with dominant expression in germ cells. We disrupted Dlec1 in mice and analysed its function in spermatogenesis and male fertility. Dlec1 deletion caused male infertility due to impaired spermatogenesis. Spermatogenesis progressed normally to step 8 spermatids in Dlec1 -/- mice, but in elongating spermatids, we observed head deformation, a shortened tail, and abnormal manchette organization. These phenotypes were similar to those of various intraflagellar transport (IFT)-associated gene-deficient sperm. In addition, DLEC1 interacted with tailless complex polypeptide 1 ring complex (TRiC) and Bardet-Biedl Syndrome (BBS) protein complex subunits, as well as α- and β-tubulin. DLEC1 expression also enhanced primary cilia formation and cilia length in A549 lung adenocarcinoma cells. These findings suggest that DLEC1 is a possible regulator of IFT and plays an essential role in sperm head and tail formation in mice.

Background: Ashbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out., Results: In the genomic analysis, 33 homozygous and 1377 heterozygous mutations in the coding sequences of the genome of MT strain were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove its ploidy, but this strain did not sporulate under the conditions tested. Heterozygous mutations detected in genes which are important for sporulation likely contribute to the sporulation deficiency of the MT strain. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. One homozygous mutation in AgILV2 gene encoding acetohydroxyacid synthase, which is also a flavoprotein in mitochondria, was found. Gene ontology (GO) enrichment analysis showed heterozygous mutations in all 22 DNA helicase genes and genes involved in oxidation-reduction process., Conclusion: This study suggests that oxidative stress and the aging of cells were involved in the riboflavin over-production in A. gossypii riboflavin over-producing mutant and provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.

A strain-designated Ys T was isolated as a filterable bacterial strain from Lake Sanaru, a brackish water lake in Hamamatsu Japan. Ys T is aerobic, Gram-negative, and slender rod shaped. Ys T grew optimally at 30 °C, pH 7.0-8.0 and without the addition of NaCl. MK-7 was the sole isoprenoid quinone. The main cellular polar lipids were phosphatidylethanolamine and unidentified amino- and polar-lipids. The predominant cellular fatty acids were C 18:0 , iso-C 14:0 and iso-C 15:0 . Phylogenetic analysis of 16S rRNA gene sequence revealed the nearest neighbours of strain Ys T to be members of the Ohtaekwangia and Chryseolinea genera with 91.2-92.1% sequence similarity. The percentages of conserved proteins (POCP) between the genomes of Ys T and related strains were less than 50%. Phenotypic analyses suggested that Ys T could not metabolize glucose and related sugars, which was discriminative from its phylogenetic relatives. We, therefore, propose a novel species in a new genus, Chryseotalea sanaruensis gen. nov., sp. nov. in the family Cytophagaceae (= JCM 30318 T  = LMG 30359 T ), based on cell size, the predominant cellular fatty acid composition, and the DNA GC content (38.9 mol%).

A moderate thermophilic dibenzofuran (DF) degrader, strain 4B1, was isolated from dioxin-contaminated soil in Vietnam under thermophilic condition. A 16S rRNA gene sequence analysis assigned the strain to genus Paenibacillus. The optimum growth temperature of strain 4B1 was 45°C with a doubling time of 2.7 h in the presence of DF as a sole carbon and energy source. The rate of its growth and DF-degradation were approximately 3-fold higher than those of a reference Paenibacillus sp. strain. The 4B1 strain degraded 89% of 1000 mg L -1 DF within 48 h cultivation at the optimum temperature. TBLASTN analysis based on its draft genome sequence revealed that this strain possessed a dbf gene cluster. The open reading frames (dbfA1A2RBC) in the cluster shared 99-100% identity with those of Paenibacillus sp. YK5, indicating that DF was likely degraded by an angular dioxygenation pathway in strain 4B1. Four genes in the dbf gene cluster (dbfA1A2BC) were partially induced by DF, which was observed by semi-quantitative RT-PCR. Quantitative PCR analysis of dbfA1 transcripts, encoding the alpha subunit of DF dioxygenase, indicated that dbfA1 was expressed 4-times higher than that of strain YK5 at 45°C. These results suggest that the faster growth and degradation of DF in strain 4B1 could be due to differences in transcriptional regulation of dbf cluster genes., (Copyright © 2019. Published by Elsevier B.V.)

Circular patterns called "fairy rings" in fields are a natural phenomenon that arises through the interaction between basidiomycete fungi and plants. Acceleration or inhibition of plant vegetative growth and the formation of mushroom fruiting bodies are both commonly observed when fairy rings form. The gene of an enzyme involved in the biosynthesis of these regulators was recently isolated in the fairy ring-forming fungus, Lepista sordida. To identify other genes involved in L. sordida fairy ring formation, we used previously generated sequence data to produce a more complete draft genome sequence for this species. Finally, we predicted the metabolic pathways of the plant growth regulators and 29 candidate enzyme-coding genes involved in fairy-ring formation based on gene annotations. Comparisons of protein coding genes among basidiomycete fungi revealed two nitric oxide synthase gene candidates that were uniquely encoded in genomes of fairy ring-forming fungi. These results provide a basis for the discovery of genes involved in fairy ring formation and for understanding the mechanisms involved in the interaction between fungi and plants. We also constructed a new web database F-RINGS ( http://bioinf.mind.meiji.ac.jp/f-rings/ ) to provide the comprehensive genomic information for L. sordida.

Green leaf volatiles (GLVs), including six-carbon (C6) aldehydes, alcohols, and esters, are formed when plant tissues are damaged. GLVs play roles in direct plant defense at wound sites, indirect plant defense via the attraction of herbivore predators, and plant-plant communication. GLV components provoke distinctive responses in their target recipients; therefore, the control of GLV composition is important for plants to appropriately manage stress responses. The reduction of C6-aldehydes into C6-alcohols is a key step in the control of GLV composition and also is important to avoid a toxic buildup of C6-aldehydes. However, the molecular mechanisms behind C6-aldehyde reduction remain poorly understood. In this study, we purified an Arabidopsis ( Arabidopsis thaliana ) NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980, named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR). CHR T-DNA knockout mutant plants displayed a normal growth phenotype; however, we observed significant suppression of C6-alcohol production following partial mechanical wounding or herbivore infestation. Our data also showed that the parasitic wasp Cotesia vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants compared with GLVs emitted from chr plants, which corresponded with reduced C6-alcohol levels in the mutant. Moreover, chr plants were more susceptible to exogenous high-dose exposure to ( Z )-3-hexenal, as indicated by their markedly lowered photosystem II activity. Our study shows that reductases play significant roles in changing GLV composition and, thus, are important in avoiding toxicity from volatile carbonyls and in the attraction of herbivore predators., (© 2018 American Society of Plant Biologists. All rights reserved.)

In response to herbivory, plants emit a blend of volatile organic compounds that includes green leaf volatiles (GLVs) and terpenoids. These volatiles are known to attract natural enemies of herbivores and are therefore considered to function as an indirect defense. Selection should favor herbivores that are able to suppress these volatile emissions, and thereby make themselves less conspicuous to natural enemies. We tested this possibility for silkworms, which were observed to leave secretions from their spinnerets while feeding on mulberry leaves. When we ablated the spinnerets of silkworms, no secretions were observed. Leaves infested by intact silkworms released smaller amounts of GLVs than leaves infested by ablated silkworms, indicating that the spinneret secretion suppressed GLV production. This difference in GLV emissions was also reflected in the behavioral response of Zenillia dolosa (Tachinidae), a parasitoid fly of silkworms. The flies laid fewer eggs when exposed to the volatiles from intact silkworm-infested leaves than when exposed to the volatiles from ablated silkworm-infested leaves. We identified a novel enzyme in the secretion from the spinneret that is responsible for the GLV suppression. The enzyme converted 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid, an intermediate in the biosynthetic pathway of GLVs, into its keto-derivative in a stereospecific manner. Taken together, this study shows that silkworms are able to feed on mulberry in a stealthy manner by suppressing GLV production with an enzyme in secretions of their spinnerets, which might be a countermeasure against induced indirect defense by mulberry plants.

Killer toxin resistant 6 (Kre6) and its paralog, suppressor of Kre null 1 (Skn1), are thought to be involved in the biosynthesis of cell wall β-(1 → 6)-D-glucan in baker's yeast, Saccharomyces cerevisiae. The Δkre6Δskn1 mutant of S. cerevisiae and other fungi shows severe growth defects due to the failure to synthesize normal cell walls. In this study, two homologs of Kre6, namely, K6LP1 (Kre6-like protein 1) and K6LP2 (Kre6-like protein 2), were identified in Aureobasidium pullulans M-2 by draft genome analysis. The Δk6lp1, Δk6lp2, and Δk6lp1Δk6lp2 mutants were generated in order to confirm the functions of the Kre6-like proteins in A. pullulans M-2. The cell morphologies of Δk6lp1 and Δk6lp1Δk6lp2 appeared to be different from those of wild type and Δk6lp2 in both their yeast and hyphal forms. The productivity of the extracellular polysaccharides, mainly composed of β-(1 → 3),(1 → 6)-D-glucan (β-glucan), of the mutants was 5.1-17.3% less than that of wild type, and the degree of branching in the extracellular β-glucan of mutants was 14.5-16.8% lower than that of wild type. This study showed that the gene disruption of Kre6-like proteins affected the cell morphology, the productivity of extracellular polysaccharides, and the structure of extracellular β-glucan, but it did not have a definite effect on the cell viability even in Δk6lp1Δk6lp2, unlike in the Δkre6Δskn1 of S. cerevisiae.

Three novel Vibrio phages were isolated from seawater in Okinawa. The Vibrio phage RYC infected Vibrio coralliilyticus SWA 07, while Vibrio phages CKB-S1 and CKB-S2 infected the coral pathogen V. coralliilyticus P1 (LMG 23696). The Vibrio phages CKB-S1 and CKB-S2 displayed head-tail structures whereas the Vibrio phage RYC showed a tailless non-enveloped capsid. All these Vibrio phages contained linear and double-stranded DNA. The whole genome sequencing revealed that Vibrio phage RYC has a larger genome size compared to Vibrio phages CKB-S1 and CKB-S2, and six tRNAs genes were found only in Vibrio phage RYC. Genome-wide comparison showed that Vibrio phage CKB-S1 was closely related, but was not identical, to Vibrio parahaemolyticus phages VP16T and VP16C. Meanwhile, the Vibrio phages RYC and CKB-S2 did not show high genome-wide similarity to any phages. These results suggest that the Vibrio phages CKB-S1, CKB-S2 and RYC are novel phages, which need further exploration, especially for their potential applications in phage therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn (II) -dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe (II) . Affinity and catalytic constants were estimated at 470 μM and 4.69 sec -1 , respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe (II) , not Zn (II) , is essential for POD activity and that Mn (II) could partially fulfill the function of Fe (II) . A mutant POD with replacement of His 183 , corresponding to one of three Zn (II) -binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe (II) as a catalytic center instead of Zn (II) . A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe (II) -dependent dioxygenase.

Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as β-primeverosides (6-O-β-D-xylopyranosyl-β-D-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here, we identified two UDP-glycosyltransferases (UGTs) from C. sinensis, UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into β-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic, and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 6'-hydroxy group of the sugar moiety of geranyl β-D-glucopyranoside, producing geranyl β-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine-141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with β-primeverosidase encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma β-primeveroside biosynthesis in C. sinensis. This information can be used to preserve tea aroma better during the manufacturing process and to investigate the mechanism of plant chemical defenses., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Systems for maintaining the viability of ejaculated sperm in the female reproductive tract are widespread among vertebrates and invertebrates. In birds, this sperm storage function is performed by specialized simple tubular invaginations called sperm storage tubules (SSTs) in the uterovaginal junction (UVJ) of the oviduct. Although the incidence and physiological reasons for sperm storage in birds have been reported extensively, the mechanisms of sperm uptake by the SSTs, sperm maintenance within the SSTs, and control of sperm release from the SSTs are poorly understood. In this study, we demonstrated that the highly conserved heat shock protein 70 (HSP70) stimulates sperm motility in vitro and also that HSP70 expressed in the UVJ may facilitate the migration of sperm released from the SSTs. Quantitative RT-PCR analysis demonstrated that the expression of HSP70 mRNA in the UVJ increases before ovulation/oviposition. Gene-specific in situ hybridization and immunohistochemical analysis with a specific antibody to HSP70 demonstrated that HSP70 is localized in the surface epithelium of the UVJ. Furthermore, injection of anti-HSP70 antibody into the vagina significantly inhibited fertilization in vivo. In addition, we found that recombinant HSP70 activates flagellar movement in the sperm and that the binding of recombinant HSP70 to the sperm surface is mediated through an interaction with voltage-dependent anion channel protein 2 (VDAC2). Our results suggest that HSP70 binds to the sperm surface by interacting with VDAC2 and activating sperm motility. This binding appears to play an important role in sperm migration within the oviduct.

The enzymatic synthesis of an α-chitin-like substance via a non-biosynthetic pathway has been achieved by transglycosylation in an aqueous system of the corresponding substrate, tri-N-acetylchitotriose [(GlcNAc)(3)] for lysozyme. A significant amount of water-insoluble product precipitated out from the reaction system. MALDI-TOFMS analysis showed that the resulting precipitate had a degree of polymerization (DP) of up to 15 from (GlcNAc)(3). Solid-state (13)C NMR analysis revealed that the resulting water-insoluble product is a chitin-like substance consisting of N-acetylglucosamine (GlcNAc) residues joined exclusively in a β-(1→4)-linked chain with stringent regio-/stereoselection. X-ray diffraction (XRD) measurement as well as (13)C NMR analysis showed that the crystal structure of synthetic product corresponds to α-chitin with a high degree of crystallinity. We propose that the multiple oligomers form an α-chitin-like substance as a result of self-assembly via oligomer-oligomer interaction when they precipitate., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ingestion of the toxic mushroom Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS-PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized N-linked sugar chains, especially Galbeta1-->4GlcNAcbeta1-->4Manbeta1-->4GlcNAcbeta1-->4GlcNAc (Type II) residues in N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Beta-glucosidase was partially purified from Rosa 'Hoh-Jun' petals. The enzyme was highly specific for such beta-D-glucopyranosides as 2-phenylethyl beta-D-glucopyranoside. The optimal activity was observed at pH 6.0 and 35 degrees C. The enzymes were composed with two proteins (160 and 155 kDa) by blue native-PAGE, and were classified in a family 1 glucosidase based on LC-MS/MS analyses.

Techniques involving fluorescein-5-isothiocyanate-conjugated gelatin injection, immunohistochemistry, and in situ reverse transcription/polymerase chain reaction (RT-PCR) revealed a close relationship between vascular endothelial growth factor (VEGF)-A-expressing cells and microvessels in the hypothalamic-pituitary axis of the rat. In situ RT-PCR clearly indicated the presence of VEGF-A mRNA-expressing cells in the pars tuberalis and in the pars distalis both at embryonic day 15.5 (E15.5) and in later developmental stages. The primary capillaries extended along the developing pars tuberalis, whereas the portal vessels penetrated into the pars distalis at E15.5 and subsequently expanded into the lobe to connect with the secondary capillary plexus, emerging in the pars distalis. At the same time, several VEGF-A-positive cells appeared in the pars distalis. These VEGF-A-positive cells were found to correspond to a portion of adrenocorticotropin (ACTH) cells by dual-staining for in situ RT-PCR and immunohistochemistry, suggesting that some ACTH cells have the potential to produce VEGF-A. Thus, the present study suggests that VEGF-A is involved in the development of the primary capillaries and in the vascularization of the pars distalis, but not in the portal vessels since the formation of portal vessels begins at E13.5, before the appearance of VEGF-A in the rostral region of the pars distalis.