Identification of quantitative trait loci for ABA responsiveness at the seedling stage associated with ABA-regulated gene expression in common wheat

Responsiveness to abscisic acid (ABA) during vegetative growth plays an important role in regulating adaptive responses to various environmental conditions, including activation of a number of ABA-responsive genes. However, the relationship between gene expression and responsiveness to ABA at the seedling stage has not been well studied in wheat. In the present study, quantitative trait locus (QTL) analysis for ABA responsiveness at the seedling stage was performed using recombinant inbred lines derived from a cross between common wheat cultivars showing different ABA responsiveness. Five QTLs were found to be significant, located on chromosomes 1B, 2A, 3A, 6D and 7B. The QTL with the greatest effect was located on chromosome 6D and explained 11.12% of the variance in ABA responsiveness. The other QTLs each accounted for approximately 5–8% of the phenotypic variation. Expression analyses of three ABA-responsive Cor/Lea genes, Wdhn13, Wrab15 and Wrab17, showed that allelic differences in QTLs on chromosomes 2A, 6D and 7B influenced expression of these genes in seedlings treated with ABA. The 3A QTL appeared to be involved in the regulatory system of Wdhn13 and Wrab15, but not Wrab17. The effects of the 2A and 6D QTLs on gene expression were relatively large. The combination of alleles at the QTLs resulted in an additive or synergistic effect on Cor/Lea expression. These results indicate that the QTLs influencing ABA responsiveness are associated with ABA-regulated gene expression and suggest that the QTL on chromosome 6D with the largest effect acts as a key regulator of ABA responses including seedling growth arrest and gene expression during the vegetative stage.     

Application of nurse culture for plant regeneration from protoplasts of Lilium japonicum thunb

Summary The regeneration of lily protoplasts isolated from suspension cells of Lilium japonicum was achieved by using the nurse culture method. The protoplasts divided only under the nurse culture application. The divided protoplasts grew into colonies and developed into visible calluses on a medium containing picloram. After the calluses were transferred to a hormone-free medium, plantlets formed from them. The highest frequency of plant regeneration was obtained on a medium containing 1 μM gibberellin 4 (GA₄). The cleaved amplified polymorphie sequences (CAPS) method was used to confirm that the regenerants were not plants that had escaped from nurse cells. We were able to transplant the plantlets to soil in pots without acclimatization, and they showed normal growth.     

Overexpression of myocilin in cultured human trabecular meshwork cells

The trabecular meshwork, a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of the trabecular meshwork is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. Its expression is known to be upregulated by glucocorticoids in trabecular meshwork cells and the altered myocilin level may be the culprit for glaucomatous conditions such as corticosteroid-induced glaucoma. In this study, we examined the influence of myocilin overexpression on the adhesion, spreading, migration, phagocytosis, and apoptosis of human trabecular meshwork cells in culture. When the myocilin expression was increased by 3- to 4-fold, the transfectants showed a dramatic loss of actin stress fibers and focal adhesions. Cell adhesion to fibronectin and spreading were also compromised. Myocilin thus appeared to have a de-adhesive activity, similar to that reported extensively with matricellular proteins. The transfected cells in addition displayed an increased sensitivity to apoptosis. These results demonstrate that overexpression of myocilin renders trabecular meshwork cells in a de-adhesive and vulnerable state. This vulnerability may be the basis for pathologic consequences in subtypes of glaucoma.     

Importance of exposed aromatic residues in chitinase B from Serratia marcescens 2170 for crystalline chitin hydrolysis

Chitinase B (ChiB) of S. marcescens has five exposed aromatic residues linearly aligned toward the catalytic cleft, Tyr481 and Trp479 in the C-terminal domain, and Trp252, Tyr240 and Phe190 in the catalytic domain. To determine the contribution of these residues to the hydrolysis of crystalline beta-chitin, site-directed mutagenesis, to replace them by alanine, was carried out. The Y481A, W479A, W252A, and Y240A mutations all decreased the binding activity and hydrolyzing activity toward beta-chitin microfibrils. Substitution of Trp residues affected the binding activity more severely than that of Tyr residues. The F190A mutation decreased neither the binding activity nor the hydrolyzing activity. None of the mutations decreased the hydrolyzing activity toward soluble substrates. These results suggest that ChiB hydrolyzes crystalline beta-chitin via a mechanism in which four exposed aromatic residues play important roles, similar to the mechanism of hydrolysis by ChiA of this bacterium, although the directions of hydrolysis of the two chitinases are opposite.     

DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity.     

NIDD, a novel DHHC-containing protein, targets neuronal nitric-oxide synthase (nNOS) to the synaptic membrane through a PDZ-dependent interaction and regulates nNOS activity

Targeting of neuronal nitric-oxide synthase (nNOS) to appropriate sites in a cell is mediated by interactions with its PDZ domain and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the central nervous system. Here we report the identification and characterization of a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank accession number AB098078), which increases nNOS enzyme activity by targeting the nNOS to the synaptic plasma membrane in a PDZ domain-dependent manner. The deduced NIDD protein consisted of 392 amino acid residues and possessed five transmembrane segments, a zinc finger DHHC domain, and a PDZ-binding motif (-EDIV) at its C-terminal tail. In vitro pull-down assays suggested that the C-terminal tail region of NIDD specifically interacted with the PDZ domain of nNOS. The PDZ dependence was confirmed by an experiment using a deletion mutant, and the interaction was further confirmed by co-sedimentation assays using COS-7 cells transfected with NIDD and nNOS. Both NIDD and nNOS were enriched in synaptosome and synaptic plasma membrane fractions and were present in the lipid raft and postsynaptic density fractions in the rat brain. Co-localization of these proteins was also observed by double staining of the proteins in cultured cortical neurons. Thus, NIDD and nNOS were co-localized in the brain, although the colocalizing regions were restricted, as indicated by the distribution of their mRNA expression. Most important, co-transfection of NIDD and nNOS increased NO-producing nNOS activity. These results suggested that NIDD plays an important role in the regulation of the NO signaling pathway at postsynaptic sites through targeting of nNOS to the postsynaptic membrane.     

Production of fertile somatic hybrid plants between Oriental hybrid lily and<Emphasis Type="Italic"> Lilium</Emphasis>×<Emphasis Type="Italic">formolongi</Emphasis>

Somatic hybridizations via electrofusion were performed in combinations of Oriental hybrid lilies (cvs. Acapulco and Shirotae) and Liliumxformolongi hort. (cv. Hakucho). Electrofusion-treated protoplasts divided only under nurse culture. The divided protoplasts grew into calli on the culture medium containing picloram, and the calli were then transferred to the hormone-free culture medium for induction of plant regeneration. The regenerants were transferred to a greenhouse, and were grown until the flower stage. In the fusion combinations of Acapulco + Hakucho and Shirotae + Hakucho, four regenerants apparently showed different morphological features compared with their parents. The results of molecular analyses by means of cleaved amplified polymorphic sequences markers and flow cytometry confirmed that these regenerants were somatic hybrid plants. Furthermore, we examined the stability of the morphological features of the hybrids in the next generations. This is the first report to describe the successful realization of Lilium somatic hybridization via protoplast fusion.     

Preparation of virosomes coated with the vesicular stomatitis virus glycoprotein as efficient gene transfer vehicles for animal cells

The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH-dependent membrane-fusion) were expected to work. Plasmid DNA (pEGFP-N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5: 1: 4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 micro m, 2 micro m, 0.65 micro m, and then 0.45 micro m). Assembly of the VSV G protein-coated liposomes (VSV G-virosomes) was performed by mixing the DNA-encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene-transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G-virosomes displayed a roughly similar level of gene-transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti-VSV G antiserum or by addition of 20 m M NH(4) Cl to transfected cultures. From these results, we assume that the virosome-mediated gene-transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.