Gibberellin 2-oxidases from seedlings of adzuki bean (Vigna angularis) show high gibberellin-binding activity in the presence of 2-oxoglutarate and Co2+

Five full-length cDNA encoding gibberellin 2-oxidases, VaGA2oxA1, VaGA2oxA2, VaGA2oxB1, VaGA2oxB2, and VaGA2oxB3, were cloned from etiolated adzuki bean (Vigna angularis cv. Dainagon) seedlings, and their enzymatic characteristics were examined using recombinant enzymes fused with glutathione S-transferase (GST). Recombinant VaGA2oxA1 (rVaGA2oxA1) and rVaGA2oxA2 showed 2beta-hydroxylation activity by converting GA1, GA4, GA9, GA20, GA4-methyl ester, and 16,17-dihydro-GA4 to the corresponding 2beta-hydroxylated gibberellins, which were identified by GC/MS. rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3 showed similar activity by converting [3H4]-16,17-dihydro-GA4 to a metabolite showing an Rf value of 16,17-dihydro-GA34. RNA-blot analysis showed that VaGA2oxA1 and VaGA2oxA2 were the major ones expressed in etiolated hypocotyls. The addition of Co2+ instead of Fe2+ to the assay medium apparently reduced the enzymatic activity, but increased the binding of [3H4]-16,17-dihydro-GA4 to rVaGA2oxA1, indicating the possibility that VaGA2oxs can be detected as gibberellin-binding proteins under certain conditions.     

Detection in situ and characterization of lignin in the<Emphasis Type="Italic"> G</Emphasis>-layer of tension wood fibres of<Emphasis Type="Italic"> Populus deltoides</Emphasis>

The occurrence of lignin in the additional gelatinous (G-) layer that differentiates in the secondary wall of hardwoods during tension wood formation has long been debated. In the present work, the ultrastructural distribution of lignin in the cell walls of normal and tension wood fibres from poplar (Populus deltoides Bartr. ex Marshall) was investigated by transmission electron microscopy using cryo-fixation–freeze-substitution in association with immunogold probes directed against typical structural motifs of lignin. The specificity of the immunological probes for condensed and non-condensed guaiacyl and syringyl interunit linkages of lignin, and their high sensitivity, allowed detection of lignin epitopes of definite chemical structures in the G-layer of tension wood fibres. Semi-quantitative distribution of the corresponding epitopes revealed the abundance of syringyl units in the G-layer. Predominating non-condensed lignin sub-structures appeared to be embedded in the crystalline cellulose matrix prevailing in the G-layer. The endwise mode of polymerization that is known to lead to these types of lignin structures appears consistent with such an organized cellulose environment. Immunochemical labelling provides the first visualization in planta of lignin structures within the G-layer of tension wood. The patterns of distribution of syringyl epitopes indicate that syringyl lignin is deposited more intensely in the later phase of fibre secondary wall assembly. The data also illustrate that syringyl lignin synthesis in tension wood fibres is under specific spatial and temporal regulation targeted differentially throughout cell wall layers.Abbreviations G-layer Gelatinous layer - G Guaiacyl monomeric unit - PATAg Periodic acid–thiocarbohydrazide–silver proteinate - S Syringyl monomeric unit     

Generative Cell Specification Requires Transcription Factors Evolutionarily Conserved in Land Plants

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Large-scale comparative mapping of the MHC class I region of predominant haplotypes in Japanese

 In order to further our understanding of major histocompatibility complex (MHC) class I gene organization, we began a comparative analysis of the large scale organization of the class I region in diverse haplotypes. For these studies, the MHC in healthy Japanese donors who have the predominant MHC haplotypes and/or HLA-A or -B alleles was examined by pulsed field gel electrophoresis and Southern analysis using probes spanning the class I region. Hybridization with probes from the HLA-A to HLA-G region revealed that individuals expressing HLA-A30, -A31, or -A33 have an approximately 70 kilobase (kb) insertion near the HLA-A gene as compared with haplotypes containing the HLA-A11 or -A26 allele. Conversely, HLA-A24-containing haplotypes appear to have an approximately 50 kb deletion from the same region. Further, it appears that chromosomes carrying closely related alleles are similar to each other in this region, consistent with their presumed evolutionary relationship. While little is known about the gene content between the HLA-A and HLA-G region, it will be interesting to examine the prospect that functional genes do in fact reside within the inserted or deleted portions, thereby raising the possibility that distinct functional differences are conferred by different haplotypes. Overall, the results reported here should contribute to furthering our understanding of the association between diseases and HLA as well as provide new insights into the evolution of the MHC. Received: 11 December 1996     

Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relation to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa

Junctional epidermolysis bullosa (JEB) is a clinically and biologically heterogeneous genodermatosis, characterized by trauma-induced blistering and healing without scarring but sometimes with skin atrophy. We investigated three unrelated patients with different JEB phenotypes. Patients 1 and 2 had generalized atrophic benign epidermolysis bullosa (GABEB), with features including skin atrophy and alopecia. Patient 3 had the localisata variant of JEB, with predominantly acral blistering and normal hair. All patients carried novel homozygous point mutations (Q1016X, R1226X, and R1303Q) in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane component; and, therefore, not only GABEB but also the localisata JEB can be a collagen XVII disorder. The nonsense mutations led to drastically reduced collagen XVII mRNA and protein levels. In contrast, the missense mutation allowed expression of abnormal collagen XVII, and epidermal extracts from that patient contained polypeptides of normal size, as well as larger aggregates. The homozygous nonsense mutations in the COL17A1 gene were consistent with the absence of the collagen from the skin and with the GABEB phenotype, whereas homozygosity for the missense mutation resulted in expression of aberrant collagen XVII and, clinically, in localisata JEB.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Immunohistochemical and histochemical evidence for the presence of noradrenaline,serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body

The immunohistochemical study revealed tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), serotonin, glutamate decarboxylase (GAD) and -aminobutyric acid (GABA) immunoreactivities in the mouse carotid body. TH and DBH immunoreactivities were found in almost all chief cells and a few ganglion cells, and in relatively numerous varicose nerve fibers of the carotid body. The histofluorescence microscopy showed catecholamine fluorescence in almost all chief cells. However, no PNMT immunoreactivity was observed in the carotid body. Serotonin, GAD and GABA immunoreactivities were also seen in almost all chief cells of the carotid body. From combined immunohistochemistry and fluorescence histochemistry, catecholamine and serotonin or catecholamine and GABA were colocalized in almost all chief cells. Thus, these findings suggest that noradrenaline, serotonin and GABA may be synthesized and co-exist in almost all chief cells of the mouse carotid body and may play roles in chemoreceptive functions.     

Simple and rapid analysis of lamotrigine, a novel antiepileptic, in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A simple and rapid method for the quantitation of concentrations of lamotrigine, a novel antiepileptic, in human serum was developed with high-performance liquid chromatography, using a solid-phase extraction technique. The mobile phase was composed of acetonitrile-10 mM phosphate buffer (pH 3.5) containing 5 mM sodium octanesulphonate (27:73, v/v), and components were detected at 265 nm. Retention times of acetanilide as an internal standard and lamotrigine were 3.4 and 10.3 min, respectively. The coefficients of variation were 3.1–4.5% and 4.4–9.8% for the within-day and between-day precision estimates, respectively. The extraction recovery of lamotrigine added to blank serum was 86–107%. The quantitation limit of lamotrigine was ca.0.2 μg/ml in 100 μl of serum. These results suggest that the method employed in this study is useful for the routine monitoring of sereum concentrations of lamotrigine in epileptic patients.     

In Planta Localization of Stilbenes within Picea abies Phloem

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.The bark of conifers has anatomically and chemically integrated defense strategies that are either constitutive (i.e. continuously produced) or inducible (i.e. activated as a response to insect or pathogen attack; Krokene, 2015). Many defense traits exist in both forms (Franceschi et al., 2005). For example, axial phloem parenchyma cells (or polyphenolic parenchyma) are critical in conifer bark defense. These cells regularly form in Pinaceae during annual phloem formation (Franceschi et al., 1998, 2000; Krekling et al., 2000; Jyske et al., 2015) but also are produced on invasion (Franceschi et al., 2005; Krokene, 2015). In Norway spruce (Picea abies) phloem, axial parenchyma forms distinctive, continuous tangential sheets across conducting (i.e. noncollapsed) and nonconducting (i.e. collapsed) tissue.Pioneering studies using microscopy with different dye agents and autofluorescence showed that the large vacuole is a special feature of the axial phloem parenchyma that contains phenolic substances (i.e. phenolic bodies; Franceschi et al., 1998). Microscopic imaging techniques also showed that polyphenolic content is highly dynamic (Franceschi et al., 1998, 2000, 2005) and changes seasonally (Krekling et al., 2000). Within the last 5 years, progress in laser microdissection (LMD) has facilitated the sampling of individual tissues and cells, providing information about the exact chemical composition of phenolic content. Li et al. (2012) used LMD to show that the axial parenchyma is the main site of phenolic accumulation in spruce bark, including that of stilbene compounds.Stilbenes are secondary metabolites that are composed of two phenol moieties linked by a C₂ bridge. These compounds are derived from the phenylpropanoid pathway, in which the last steps of biosynthesis are catalyzed by stilbene synthase (Chong et al., 2009). There is increasing interest in these antioxidant, antibacterial, and antiinflammatory compounds for use in healthy human diets, therapeutic approaches, and as protective agents in materials sciences (Shibutani et al., 2004; Metsämuuronen and Siren, 2014; Reinisalo et al., 2015; Hedenström et al., 2016; Sirerol et al., 2016). The tetrahydroxystilbene glucosides trans-astringin (3,3ʹ,4ʹ,5-tetrahydroxystilbene 3-O-β-d-glucoside) and trans-isorhapontin (3,4ʹ,5-trihydroxy-3ʹ-methoxystilbene 3-O-β-d-glucoside) are the most abundant constitutive stilbene compounds of Norway spruce, while the trihydroxystilbene glucoside trans-piceid (resveratrol 3-O-β-glucoside) and stilbene aglycons (i.e. without the sugar moiety) are less abundant. Stilbene synthesis in spruce probably proceeds through the formation of resveratrol (i.e. aglycon of piceid) followed by further modifications (i.e. hydroxylation, O-methylation, and O-glycosylation) to yield tetrahydroxystilbene glucosides (Hammerbacher et al., 2011). Stilbenes are assumed to provide protection against a wide variety of environmental stressors (Franceschi et al., 2005; Witzell and Martin, 2008; Chong et al., 2009). Stilbenes appear to contribute to antifungal defense in spruce (Hammerbacher et al., 2011, 2013). The fungal inoculation of spruce bark with the blue-stain fungus Endoconidiophora polonica (previously named Ceratocystis polonica; de Beer et al., 2014) causes astringin levels to decrease, in parallel with increasing dimeric stilbene glucoside levels in the LMD-isolated axial phloem parenchyma (Li et al., 2012) or increasing levels of corresponding aglycons in bulk tissue (Viiri et al., 2001). During the annual formation of phloem in Norway spruce, the accumulation of stilbene glucosides inside the newest, LMD-isolated phloem ring is preceded by the formation and cellular development of a new band of axial parenchyma (Jyske et al., 2015). These observations strongly indicate that the inducible and constitutive stilbene compounds of spruce phloem are both stored and synthesized in the axial parenchyma.New mass spectrometry imaging techniques provide significant improvements in the mapping of plant metabolites (Briggs and Seah, 1993; Vickerman and Briggs, 2001; Burrell et al., 2007; Cha et al., 2008; Lee et al., 2012; Bjarnholt et al., 2014; Aoki et al., 2016). To elucidate the synthesis, distribution, and metabolism of secondary plant metabolites, it is essential to gather positional information about them in a living state, as pretreatment of specimens, such as drying, may change the distribution and concentration features of soluble chemicals (Metzner et al., 2008; Li et al., 2012; Kuroda et al., 2013). In this study, we used a unique system of time-of-flight secondary ion mass spectrometry and scanning electron microscopy connected with a cryo-shuttle (cryo-TOF-SIMS/SEM) to study the localization and accumulation patterns of stilbenes within cells and tissues of phloem. This system has been developed to study chemical distributions at high-spatial resolution (1 µm) directly from the surfaces of plant specimens in a frozen-hydrated state (i.e. in planta) representing living tissues (Kuroda et al., 2013; Aoki et al., 2016). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) directly detects organic and inorganic compounds on the specimen surface over a broad mass-to-charge ratio (m/z) range by mass spectrometry with high chemical sensitivity. Specimen surface morphology is visualized by the detection of total secondary ion content. The quality of cellular integrity may be further observed by scanning electron microscopy connected with a cryo-shuttle (cryo-SEM) imaging of the frozen surface of the same specimen. The cryo-TOF-SIMS/SEM system has still rarely been applied to the analysis of plant physiology (Metzner et al., 2008, 2010; Iijima et al., 2011; Kuroda et al., 2013; Aoki et al., 2016).Mass spectrometer imaging techniques consist of an ionizer and a mass analyzer. In the TOF-SIMS system, secondary ion mass spectrometry is used as an ionizer and time-of-flight as a mass analyzer. In another mainstream imaging mass spectrometry technique, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption/ionization is used as ionizer. Compared with TOF-SIMS, MALDI-MS is more quantitative and has high-M_r acceptance, but the resolution of MALDI-MS is not high enough for cell-level detection (Aoki et al., 2016). Instead, the spatial resolution of TOF-SIMS is superior to focus on cell functions. The disadvantage of TOF-SIMS is that the ionization and fragmentation phenomenon may be affected by the matrix effect, causing some degree of uncertainty. However, when time-of-flight secondary ion mass spectrometry connected with a cryo-shuttle (cryo-TOF-SIMS) is used in combination with quantitative gas chromatography, it is very powerful to study the positional and temporal distributions of metabolites within living plants.To complement TOF-SIMS analysis, we applied quantitative chemical microanalysis methods to study the amounts of stilbene glucosides and to correlate those with the amounts of total extractives, monosaccharides and disaccharides, and water across phloem and bark. The methods include tangential cryo-sectioning of tissues and their chemical microanalysis by gas chromatography with flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).To combine the chemical information with phloem morphology, the cellular and subcellular features of the axial phloem parenchyma were analyzed by three-dimensional (3D) synchrotron radiation microtomography (µCT). µCT is a prominent tool that has gained popularity for 3D analysis of xylem structure and physiology (Brodersen, 2013; Cochard et al., 2015), but only recently has it been applied to the 3D analysis of phloem (Jyske et al., 2015). This method offers advantages over traditional light microscopic approaches, as high-throughput data at the submicrometer level can be produced from significantly larger tissue volumes. The data allow for representative volumetric analysis of cellular distributions along with 3D visualization of subcellular features.In this study, we used a novel combination of cutting-edge techniques to analyze in parallel (1) in planta cellular localization and accumulation of stilbene glucosides across phloem and bark by semiquantitative cryo-TOF-SIMS/SEM; (2) tissue-level quantitative amounts of stilbene glucosides, total extractives, and monosaccharides and disaccharides across phloem and bark by tangential cryo-sectioning and GC-FID and GC-MS; (3) 3D cell abundance distributions across phloem and bark by µCT; and (4) variation in water content across phloem and bark (Fig. 1).Open in a separate windowFigure 1.Schematic presentation of the specimen structure and preparation for different analyses. Sample blocks were taken from living tree stem (A) or stem discs (B) at 1.3 m on the stem. The blocks (C) containing outer bark (periderm), phloem, cambium, and part of the outermost xylem ring (D; transverse view of phloem and bark) were further divided into subblocks (1–3; C and E). Subblocks 1 and 2 were quick frozen, and subblock 3 was fixed chemically. Subblock 1 was used for the direct chemical mapping of stilbenes across the phloem from the cambium to the outer bark (i.e. semiquantitative analysis of stilbene localization and accumulation across transverse and radial surfaces [purple] of the tissue block by TOF-SIMS; E-1). To obtain quantitative data on the amounts of stilbenes, other extractives, and carbohydrates across phloem and bark, tangential cryo-sections (250 or 450 µm each; cut slices illustrated with purple in E-2) were cut across subblock 2 and directed for chemical microanalysis by GC-FID (E-2). Subblock 3 was divided into four to six zones, and from each zone, small cuboids (illustrated with purple in E-3) were cut and directed for morphological analysis of phloem by phase-contrast µCT (E-3). Water content across the phloem and bark was analyzed from separate fresh blocks, which were further cut tangentially into thin sections. Black arrows indicate the radial direction from the cambium toward the outer bark. Purple areas show the analyzed locations of each subblock (E). Note that schematic drawings are not to scale.