Acetylcholinesterase (AChE) has been increasingly recognized in plants by indirect evidence of its activity. Here, we report purification and cloning of AChE from maize (Zea mays), thus providing to our knowledge the first direct evidence of the AChE molecule in plants. AChE was identified as a mixture of disulfide-and noncovalently linked 88-kD homodimers consisting of 42-to 44-kD polypeptides. The AChE hydrolyzed acetylthiocholine and propyonylthiocholine, but not S-butyrylthiocholine, and the AChE-specific inhibitor neostigmine bromide competitively inhibited its activity, implying that maize AChE functions in a similar manner as the animal enzyme. However, kinetic analyses indicated that maize AChE showed a lower affinity to substrates and inhibitors than animal AChE. The full-length cDNA of maize AChE gene is 1,471 nucleotides, which encode a protein having 394 residues, including a signal peptide. The deduced amino acid sequence exhibited no apparent similarity with that of the animal enzyme, although the catalytic triad was the same as in the animal AChE. In silico screening indicated that maize AChE homologs are widely distributed in plants but not in animals. These findings lead us to propose that the AChE family, as found here, comprises a novel family of the enzymes that is specifically distributed in the plant kingdom.

Root-associated microbial communities are very important in the adaptation of halophytes to coastal environments. However, little has been reported on microbial community structures related to halophytes, or on comparisons of their compositions among halophytic plant species. Here, we studied the diversity and community structure of both rhizosphere and root endosphere bacteria in two halophytic plants: Glaux maritima and Salicornia europaea. We sampled the rhizosphere, the root endosphere, and bulk control soil samples, and performed bacterial 16S rRNA sequencing using the Illumina MiSeq platform to characterize the bacterial community diversities in the rhizosphere and root endosphere of both halophytes. Among the G. maritima samples, the richness and diversity of bacteria in the rhizosphere were higher than those in the root endosphere but were lower than those of the bulk soil. In contrast for S. europaea, the bulk soil, the rhizosphere, and the root endosphere all had similar bacterial richness and diversity. The number of unique operational taxonomic units within the root endosphere, the rhizosphere, and the bulk soil were 181, 366, and 924 in G. maritima and 126, 416, and 596 in S. europaea, respectively, implying habitat-specific patterns for each halophyte. In total, 35 phyla and 566 genera were identified. The dominant phyla across all samples were Proteobacteria and Bacteroidetes. Actinobacteria was extremely abundant in the root endosphere from G. maritima. Beneficial bacterial genera were enriched in the root endosphere and rhizosphere in both halophytes. Rhizobium, Actinoplanes, and Marinomonas were highly abundant in G. maritima, whereas Sulfurimonas and Coleofasciculus were highly abundant in S. europaea. A principal coordinate analysis demonstrated significant differences in the microbiota composition associated with the plant species and type of sample. These results strongly indicate that there are clear differences in bacterial community structure and diversity between G. maritima and S. europaea. This is the first report to characterize the root microbiome of G. maritima, and to compare the diversity and community structure of rhizosphere and root endosphere bacteria between G. maritima and S. europaea.

A new ␤1,4-N-acetylglucosaminyltransferase (GnT) which involves in branch formation of Asn-linked complex-type sugar chains has been purified 224,000-fold from bovine small intestine. This enzyme requires divalent cations, such as Mn 2؉, and catalyzes the transfer of GlcNAc from UDP-GlcNAc to biantennary oligosaccharide and produces triantennary oligosaccharide with the ␤1-4-linked GlcNAc residue on the Man␣1-3 arm. The purified enzyme shows a single band of M r 58,000 and behaves as a monomer. The substrate specificity demonstrated that the ␤1-2-linked GlcNAc residue on the Man␣1-3 arm (GnT-I product) is essential for the enzyme activity. ␤1-4-Galactosylaion to this essential ␤1-2-linked GlcNAc residue or N-acetylglucosaminylation to the ␤-linked Man residue (bisecting GlcNAc, GnT-III product) blocks the enzyme action, while ␤1-6-Nacetylglucosaminylation to the Man␣1-6 arm (GnT-V product) increases the transfer. Based on these findings, we conclude that the purified enzyme is UDP-Nacetylglucosamine:␣-3-D-mannoside ␤-1,4-N-acetylglucosaminyltransferase IV (GnT-IV), that has been a missing link on biosynthesis of complex-type sugar chains.The complex-type of oligosaccharides are synthesized through elongation by glycosyltransferases after trimming of the precursor oligosaccharides transferred to proteins in the endoplasmic reticulum. N-Acetylglucosaminyltransferases (GnTs) 1 take part in the formation of branches in the biosynthesis of complex-type sugar chains. In vertebrates, six GnTs, designated as GnT-I to -VI, which catalyze the transfer of GlcNAc to the core mannose residues of Asn-linked sugar chains, have been identified (1),

A novel lectin was isolated from mycelia of the basidiomycete Pleurotus cornucopiae grown on solid medium. The lectin was purified to homogeneity by mucin-Sepharose affinity chromatography. The molecular mass of the lectin was 40 kDa under reducing conditions, but the subunits were polymerized through disulfide bridges under physiological conditions. Hemagglutinating activity of this lectin was completely inhibited by 2-mercaptoethanol, indicating that the multimer is active. The activity was also inhibited by EDTA, and restored by CaCl 2 . N-Acetyl-D-galactosamine was the most potent hapten inhibitor. N-terminal amino acid sequence analysis revealed that the mycelial lectin was different from the fruit body lectin of this organism. The mycelial lectin appeared prior to fruit body formation and disappeared during the formation of fruit bodies. The lectin was localized on the surface of solid-medium-grown mycelia, and only dikaryotic, and not monokaryotic, mycelia produced the lectin. These results suggest that the appearance of this lectin is associated with fruit body formation.Lectins are carbohydrate-binding proteins that are found in various organisms (21). Although there have been several reports of fungal lectins found in fruit bodies (8-10, 13, 18, 23, 24), there are few reports of mycelial lectins (17), and their physiological functions in nature have not been completely explained yet. We have found a new lectin in the fruit body of Pleurotus cornucopiae and characterized it (8, 24). This lectin was not contained in vegetatively growing mycelia in liquid medium, but it appeared after the onset of fruit body formation on solid medium (8). In the course of studies on the function of this lectin, we found and established two strains of P. cornucopiae, named KC-1 and KC-2, with respect to the lectin content in their fruit bodies (14,15).In this study, we found that mycelia of P. cornucopiae grown on solid medium exhibit hemagglutinating activity and that the active substance is different from the lectin found in the fruit body. We describe here the purification and properties as well as localization and developmental stage-specific expression of this novel mycelial lectin, suggesting that the lectin participates in the process of fruit body formation in this organism. The results presented in this paper together with those of our other studies (8, 24) demonstrate that two lectins are produced in P. cornucopiae in a developmental stage-specific manner. MATERIALS AND METHODSOrganisms. Two strains of P. cornucopiae, KC-1 and KC-2, were studied. The former contains the fruit body lectin (PCL-F), but the latter does not (15,24). Dikaryotic mycelia of these strains were maintained on agar slants of yeast extract-malt extract-glucose medium (8). Monokaryotic mycelia were obtained by single-spore isolation.Fruit body formation. Dikaryotic mycelia on the slant were first grown in liquid yeast extract-malt extract-glucose medium at 28ЊC for 7 days with shaking (stage 0). The liquid-medium-grown mycelia were inoculate...

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