Tensile-shear properties of steel-Al adhesively bonded dissimilar joints and the effect of Al plate thickness.

Lap shear dissimilar joints between aluminium (Al) alloy, A6061-T6, and stainless steel, type 304, were fabricated by adhesive bonding. Three Al plates with different thicknesses were used to investigate the effect of the Al plate thickness on the tensile-shear properties, namely the effect of bending stiffness of Al plates. The maximum tensile-shear loads increased with increasing Al plate thicknesses. The fracture through the adhesive layer (cohesive fracture) occurred when the Al plate was the thickest, while the interface fracture between Al plate and adhesive layer appeared on the fracture surface with decreasing Al plate thickness. Fatigue strengths also increased with increasing Al plate thickness. When the fatigue strengths were normalized by the tensile strengths, the effect of the plate thickness became negligible. FEM analyses revealed that the stress concentration at the edge of adhesive on Al side decreased with increasing Al plate thickness, which could be related to the dependence of tensile and fatigue properties on the Al plate thickness.

Omics Profiles of Non-transgenic Scion Grafted on Transgenic RdDM Rootstock.

Grafting of commercial varieties onto transgenic stress-tolerant rootstocks is attractive approach, because fruit from the non-transgenic plant body does not contain foreign genes. RNA silencing can modulate gene expression and protect host plants from viruses and insects, and small RNAs (sRNAs), key molecules of RNA silencing, can move systemically. Here, to evaluate the safety of foods obtained from sRNA-recipient plant bodies, we investigated the effects of rootstock-derived sRNAs involved in mediating RNA-directed DNA methylation (RdDM) on non-transgenic scions. We used tobacco rootstocks showing RdDM against the cauliflower mosaic virus (CaMV) 35S promoter. When scions harboring CaMV 35S promoter sequence were grafted onto RdDM-inducing rootstocks, we found that RdDM-inducing sRNAs were only weakly transported from the rootstocks to the scion, and we observed a low level of DNA methylation of the CaMV 35S promoter in the scion. Next, wild-type (WT) tobacco scions were grafted onto RdDM-inducing rootstocks (designated NT) or WT rootstocks (designated NN), and scion leaves were subjected to multi-omics analyses. Our transcriptomic analysis detected 55 differentially expressed genes between the NT and NN samples. A principal component analysis of proteome profiles showed no significant differences. In the positive and negative modes of LC-ESI-MS and GC-EI-MS analyses, we found a large overlap between the metabolomic clusters of the NT and NN samples. In contrast, the negative mode of a LC-ESI-MS analysis showed separation of clusters of NT and NN metabolites, and we detected 6 peak groups that significantly differed. In conclusion, we found that grafting onto RdDM-inducing rootstocks caused a low-level transmission of sRNAs, resulting in limited DNA methylation in the scion. However, the causal relationships between sRNA transmission and the very slight changes in the transcriptomic and metabolomic profiles of the scions remains unclear. The safety assessment points for grafting with RdDM rootstocks are discussed.

Effect of Transgenic Rootstock Grafting on the Omics Profiles in Tomato.

Grafting of non-transgenic scion onto genetically modified (GM) rootstocks provides superior agronomic traits in the GM rootstock, and excellent fruits can be produced for consumption. In such grafted plants, the scion does not contain any foreign genes, but the fruit itself is likely to be influenced directly or indirectly by the foreign genes in the rootstock. Before market release of such fruit products, the effects of grafting onto GM rootstocks should be determined from the perspective of safety use. Here, we evaluated the effects of a transgene encoding ß-glucuronidase (GUS) on the grafted tomato fruits as a model case. An edible tomato cultivar, Stella Mini Tomato, was grafted onto GM Micro-Tom tomato plants that had been transformed with the GUS gene. The grafted plants showed no difference in their fruit development rate and fresh weight regardless of the presence or absence of the GUS gene in the rootstock. The fruit samples were subjected to transcriptome (NGS-illumina), proteome (shotgun LC-MS/MS), metabolome (LC-ESI-MS and GC-EI-MS), and general food ingredient analyses. In addition, differentially detected items were identified between the grafted plants onto rootstocks with or without transgenes (more than two-fold). The transcriptome analysis detected approximately 18,500 expressed genes on average, and only 6 genes were identified as differentially expressed. Principal component analysis of 2,442 peaks for peptides in proteome profiles showed no significant differences. In the LC-ESI-MS and GC-EI-MS analyses, a total of 93 peak groups and 114 peak groups were identified, respectively, and only 2 peak groups showed more than two-fold differences. The general food ingredient analysis showed no significant differences in the fruits of Stella scions between GM and non-GM Micro-Tom rootstocks. These multiple omics data showed that grafting on the rootstock harboring the GUS transgene did not induce any genetic or metabolic variation in the scion.

NaCl dependent production of coniferin in Alluaudiopsis marnieriana suspension cultured cells.

A stable salt-tolerant cell-suspension culture of Alluaudiopsis marnieriana was established, and intracellular compounds that accumulated under salt-stress conditions were investigated. HPLC/MS, and NMR analyses indicated that enhanced accumulation of coniferin was found during the growth phase in medium containing 150 mM NaCl. Coniferin or its derivatives may play an important role in salt-tolerance mechanisms in this plant.

UHPLC/MS and NMR-Based Metabolomic Analysis of Dried Water Extract of Citrus-Type Crude Drugs.

Citrus-type crude drugs (CCDs) are commonly used to formulate decoctions in Kampo formula (traditional Japanese medicine). Our previous study reported metabolomic analyses for differentiation of the methanol extracts of Citrus-type crude drugs (CCDs) using ultra-HPLC (UHPLC)/MS, and 13C- and 1H-NMR. The present study expanded the scope of its application by analyzing four CCD water extracts (Kijitsu, Tohi, Chimpi, and Kippi); these CCDs are usually used as decoction ingredients in the Kampo formula. A principal component analysis score plot of processed UPLC/MS and NMR analysis data indicated that the CCD water extracts could be classified into three groups. The loading plots showed that naringin and neohesperidin were the distinguishing components. Three primary metabolites, α-glucose, ß-glucose, and sucrose were identified as distinguishing compounds by NMR spectroscopy. During the preparation of CCD dry extracts, some compounds volatilized or decomposed. Consequently, fewer compounds were detected than in our previous studies using methanol extract. However, these results suggested that the combined NMR- and LC/MS-based metabolomics can discriminate crude drugs in dried water extracts of CCDs.

Esterified carotenoids are synthesized in petals of carnation (Dianthus caryophyllus) and accumulate in differentiated chromoplasts.

Although yellow and orange petal colors are derived from carotenoids in many plant species, this has not yet been demonstrated for the order Caryophyllales, which includes carnations. Here, we identified a carnation cultivar with pale yellow flowers that accumulated carotenoids in petals. Additionally, some xanthophyll compounds were esterified, as is the case for yellow flowers in other plant species. Ultrastructural analysis showed that chromoplasts with numerous plastoglobules, in which flower-specific carotenoids accumulate, were present in the pale yellow petals. RNA-seq and RT-qPCR analyses indicated that the expression levels of genes for carotenoid biosynthesis and esterification in pale yellow and pink petals (that accumulate small amounts of carotenoids) were similar or lower than in green petals (that accumulate substantial amounts of carotenoids) and white petals (that accumulate extremely low levels of carotenoids). Pale yellow and pink petals had a considerably lower level of expression of genes for carotenoid degradation than white petals, suggesting that reduced degradation activity caused accumulation of carotenoids. Our results indicate that some carnation cultivars can synthesize and accumulate esterified carotenoids. By manipulating the rate of biosynthesis and esterification of carotenoids in these cultivars, it should be feasible to produce novel carnation cultivars with vivid yellow flowers.

Structural Analysis of Polygalaxanthones, C-Glucosyl Xanthones of Polygala tenuifolia Roots.

Polygalaxanthone III, a xanthone glycoside that is a major constituent of "Polygala Root" (Polygala tenuifolia roots, Onji in the Japanese Pharmacopoeia), has been used as a standard in the quality control of crude drugs. However, we previously noted differences in the chromatographic properties of one of three samples of polygalaxanthone III. Therefore, standardization of the standard itself is extremely important. The structures of three standard samples commercially available as polygalaxanthone III were characterized by LC/MS and NMR. LC/MS analysis revealed that two molecular types exist. Both types are chromatographically separable but have an identical mass number with distinguishable MS/MS spectra. One dimensional (1D)-NMR analyses demonstrated that both had the same xanthone moiety and heteronuclear multiple bond correlation (HMBC) analyses revealed that they are structural isomers at the connecting position of glucose to apiose 1-position. Consequently, the isomers were identified as polygalaxanthone III and its regioisomer, polygalaxanthone XI. Based on the findings, we recommend using the LC-MS/MS detection method, which discriminates polygalaxanthone III and XI, to confirm the quality of the standard.

Development and evaluation of novel salt-tolerant Eucalyptus trees by molecular breeding using an RNA-Binding-Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.).

The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA-Binding-Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened-house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants' acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high-stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non-transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate-stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non-transgenic status survived the 70 mM NaCl treatment, but after 6-month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non-transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non-transgenic plants after 15-week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.

Carnation I locus contains two chalcone isomerase genes involved in orange flower coloration.

Carnations carrying a recessive I gene show accumulation of the yellow pigment chalcononaringenin 2'-glucoside (Ch2'G) in their flowers, whereas those with a dominant I gene do accumulation the red pigment, anthocyanin. Although this metabolic alternative at the I gene could explain yellow and red flower phenotypes, it does not explain the development of orange flower phenotypes which result from the simultaneous accumulation of both Ch2'G and anthocyanin. The carnation whole genome sequencing project recently revealed that two chalcone isomerase genes are present, one that is consistent with the I gene (Dca60979) and another (Dca60978) that had not been characterized. Here, we demonstrate that Dca60979 shows a high level of gene expression and strong enzyme activity in plants with a red flower phenotype; however, functional Dca60979 transcripts are not detected in plants with an orange flower phenotype because of a dTdic1 insertion event. Dca60978 was expressed at a low level and showed a low level of enzyme activity in plants, which could catalyze a part of chalcone to naringenin to advance anthocyanin synthesis but the other part remained to be catalyzed chalcone to Ch2'G by chalcone 2'-glucosyltransferase, resulting in accumulation of anthocyanin and Ch2'G simultaneously to give orange color.

Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation.

In a previous study, two genes responsible for white flower phenotypes in carnation were identified. These genes encoded enzymes involved in anthocyanin synthesis, namely, flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), and showed reduced expression in the white flower phenotypes. Here, we identify another candidate gene for white phenotype in carnation flowers using an RNA-seq analysis followed by RT-PCR. This candidate gene encodes a transcriptional regulatory factor of the basic helix-loop-helix (bHLH) type. In the cultivar examined here, both F3H and DFR genes produced active enzyme proteins; however, expression of DFR and of genes for enzymes involved in the downstream anthocyanin synthetic pathway from DFR was repressed in the absence of bHLH expression. Occasionally, flowers of the white flowered cultivar used here have red speckles and stripes on the white petals. We found that expression of bHLH occurred in these red petal segments and induced expression of DFR and the following downstream enzymes. Our results indicate that a member of the bHLH superfamily is another gene involved in anthocyanin synthesis in addition to structural genes encoding enzymes.

Synthesis of alkyne-tagged and biotin-tagged Sortin1 as novel photoaffinity probes.

Sortin1 is an inhibitor of vesicular biogenesis and transport, which is shared among eukaryotes and plants with an unknown mode of action. Toward exploration of its target proteins, we developed alkyne as well as biotin conjugated photoaffinity probes derived from Sortin1. Due to the presence of phenylketone moiety, Sortin1 was anticipated to serve as a photoreactive group in a similar manner to a commonly used photoreactive group, benzophenone. The core structure based on 5-oxo-1,4-dihydroindenopyridine was constructed in one step using three-component Hantzsch dihydropyridine synthesis. We demonstrated that Sortin1 displayed photocrosslinking reactivity against a model binding protein, which would be useful for capturing and detecting binding proteins.

Fluorescence Coupled with Macro and Microscopic Examinations of Morphological Phenotype Give Key Characteristics for Identification of Crude Drugs Derived from Scorpions.

Microscopic examination of crude drug components has been the traditional method to identify the origin of biological materials. For the identification of components in a given mixture via microscopy, standard reference photographs of fragments derived from different organs and tissues of individual species are required. In addition to these reference photographs, a highly observant eye is needed to compare the morphological characteristics observed under the microscope with those of the references and to then identify the origin of the materials. Therefore, if other indexes are available to be coupled with microscope examination, the accuracy of identification would be significantly improved. Here, we prepared standard reference photographs for microscopic examination to identify powdered and fragmented materials in the crude drug "Quanxie" derived from individual organs of dried scorpion (Buthus martensii KARSCH). Since a remarkable characteristic of scorpion bodies is that they fluoresce under UV light, two methods to identify "Quanxie" were established, including fluorescence fingerprint analysis and microscopic fluorescent luminance imaging analysis. In the former, at least 0.1 g of powered materials was used, which could be recovered after the measurement, and in the latter, only small amounts of powders were used for microscopic examinations. Both methods could distinguish powders of "Quanxie" from those of other micro-morphologically similar crude drugs, namely, "Chantui," "Sangpiaoxiao," and "Jiangcan." The combination of these methods should improve the swiftness and accuracy of "Quanxie" identification.

A double knockout mutant of acyl-glucose-dependent anthocyanin glucosyltransferase genes in Delphinium grandiflorum.

Blue coloration in delphinium flowers arises from 7-polyacylated anthocyanins which are modified alternately with acyl and glucosyl residues at the 7 position of the aglycone. Previously, we identified two independent genes for acyl-glucose-dependent anthocyanin 7-(6-(p-hydroxybenzoyl)-glucoside) glucosyltransferases (AA7BG-GT); recombinant proteins from the two cDNAs were produced in Escherichia coli and showed AA7BG-GT activity in vitro. Here, a double knockout mutant of both genes was found to lack modification of the second glucosyl residue following further acyl and glucosyl modifications. Both genes in the double mutant had nucleotide sequence changes and deletions that disrupted their transcripts and caused loss of AA7BG-GT activity in sepals. These results provide genetic confirmation that both genes are responsible for AA7BG-GT enzyme activity.

Identification of flavonoid 3'-hydroxylase in the yellow flower of Delphinium zalil.

The flowers of delphinium cultivars owe their coloration to anthocyanins such as delphinidin or pelargonidin derivatives. To date, no delphinium cultivars have been found with red flowers due to the presence of cyanidin derivatives. This suggests that delphiniums do not have cyanidin biosynthesis ability because of the loss of function of flavonoid 3' hydroxylase (F3'H). Here, we show that the wild delphinium species Delphinium zalil (synonym semibarbatum) can accumulate quercetin 3-glucosides in its sepals, presumably through F3'H activity. We isolated F3'H cDNA from D. zalil (DzF3'H) and produced a recombinant enzyme from a yeast transformant. The recombinant DzF3'H protein could convert naringenin, apigenin, dihydrokaempferol and kaempferol to eriodictyol, luteolin, dihydroquercetin and quercetin, respectively. An expression analysis confirmed that blue flowered D. grandiflorum does not express F3'H, and also showed that flavonoid 3',5'-hydroxylase and anthocyanidin synthase do not function in D. zalil sepals. DzF3'H can act as a flavonoid hydroxylase to produce cyanidin accumulation. The introduction of the DzF3'H gene into other delphinium species by conventional breeding may enable development of cultivars with novel flower colors.

Detection of Nicotiana tabacum Leaf Contamination in Pharmaceutical Products.

Nicotiana tabacum (Solanaceae) is the only species whose leaves can be legally marketed as tobacco according to the Japanese Tobacco Business Act. Nicotine, a major alkaloid produced by N. tabacum leaves, is regulated in pharmaceuticals by the Japanese Pharmaceutical Affairs Law. However, the use of N. tabacum stems as an excipient in pharmaceuticals is permitted, because these contained only a small amount of nicotine. Recently, several reports showed that a substantial amount of nicotine was detected in an OTC pharmaceutical product, in which N. tabacum stems were used as an excipient. Therefore, products containing N. tabacum stems could be contaminated with the leaf material. In the present study, we established a method to detect contamination of N. tabacum stem materials with its leaves, using microscopy to obtain standard reference microphotographs for identification. Cultivated N. tabacum stems and leaves, commercial cigarette leaves, and N. tabacum tissue imported as excipient material were used for preparing the microphotographs. The characteristic N. tabacum leaf structures found in the powdered fragments included: epidermal cells with sinuous anticlinal cell walls, hairs, mesophyll parenchyma with crystalized calcium oxalate (calciphytoliths), and branching vascular bundles derived from reticulate net-veins. A comparison of the microscopic characteristics of an OTC powder with those from the standard reference microphotographs was an effective method for N. tabacum stem and leaf identification. Thus, we evaluated the powdered pharmaceutical product containing N. tabacum stem tissue and Hydrangea serrata (Hydrangeaceae) leaf tissue as excipients, and confirmed the presence of N. tabacum leaf material.

The role of acyl-glucose in anthocyanin modifications.

Higher plants can produce a wide variety of anthocyanin molecules through modification of the six common anthocyanin aglycons that they present. Thus, hydrophilic anthocyanin molecules can be formed and stabilized by glycosylation and acylation. Two types of glycosyltransferase (GT) and acyltransferase (AT) have been identified, namely cytoplasmic GT and AT and vacuolar GT and AT. Cytoplasmic GT and AT utilize UDP-sugar and acyl-CoA as donor molecules, respectively, whereas both vacuolar GT and AT use acyl-glucoses as donor molecules. In carnation plants, vacuolar GT uses aromatic acyl-glucoses as the glucose donor in vivo; independently, vacuolar AT uses malylglucose, an aliphatic acyl-glucose, as the acyl-donor. In delphinium and Arabidopsis, p-hydroxybenzoylglucose and sinapoylglucose are used in vivo as bi-functional donor molecules by vacuolar GT and AT, respectively. The evolution of these enzymes has allowed delphinium and Arabidopsis to utilize unique donor molecules for production of highly modified anthocyanins.

Transposon-mediated mutation of CYP76AD3 affects betalain synthesis and produces variegated flowers in four o'clock (Mirabilis jalapa).

The variegated flower colors of many plant species have been shown to result from the insertion or excision of transposable elements into genes that encode enzymes involved in anthocyanin synthesis. To date, however, it has not been established whether this phenomenon is responsible for the variegation produced by other pigments such as betalains. During betalain synthesis in red beet, the enzyme CYP76AD1 catalyzes the conversion of L-dihydroxyphenylalanine (DOPA) to cyclo-DOPA. RNA sequencing (RNA-seq) analysis indicated that the homologous gene in four o'clock (Mirabilis jalapa) is CYP76AD3. Here, we show that in four o'clock with red perianths, the CYP76AD3 gene consists of one intron and two exons; however, in a mutant with a perianth showing red variegation on a yellow background, a transposable element, dTmj1, had been excised from the intron. This is the first report that a transposition event affecting a gene encoding an enzyme for betalain synthesis can result in a variegated flower phenotype.

Identification of the glucosyltransferase gene that supplies the p-hydroxybenzoyl-glucose for 7-polyacylation of anthocyanin in delphinium.

In delphiniums (Delphinium grandiflorum), blue flowers are produced by the presence of 7-polyacylated anthocyanins. The polyacyl moiety is composed of glucose and p-hydroxybenzoic acid (pHBA). The 7-polyacylation of anthocyanin has been shown to be catalysed by two different enzymes, a glucosyltransferase and an acyltransferase; both enzymes utilize p-hydroxybenzoyl-glucose (pHBG) as a bi-functional (Zwitter) donor. To date, however, the enzyme that synthesizes pHBG and the gene that encodes it have not been elucidated. Here, five delphinium cultivars were investigated and found to show reduced or undetectable 7-polyacylation activity; these cultivars synthesized delphinidin 3-O-rutinoside (Dp3R) to produce mauve sepals. One cultivar showed a deficiency for the acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AA7GT) necessary for mediating the first step of 7-polyacylation. The other four cultivars showed both AA7GT activity and DgAA7GT expression; nevertheless, pHBG accumulation was significantly reduced compared with wild-type cultivars, whereas p-glucosyl-oxybenzoic acid (pGBA) was accumulated. Three candidate cDNAs encoding a UDP-glucose-dependent pHBA glucosyltransferase (pHBAGT) were identified. A phylogenetic analysis of DgpHBAGT amino acid sequences showed a close relationship with UGTs that act in acyl-glucose synthesis in other plant species. Recombinant DgpHBAGT protein synthesized pHBG and had a high preference for pHBA in vitro. Mutant cultivars accumulating pGBA had very low expression of DgpHBAGT, whereas expression during the development of sepals and tissues in a wild cultivar showed a close correlation to the level of accumulation of pHBG. These results support the conclusion that DgpHBAGT is responsible for in vivo synthesis of pHBG in delphiniums.

Differential analysis of protein expression in RNA-binding-protein transgenic and parental rice seeds cultivated under salt stress.

Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds.

Sequence analysis of the genome of carnation (Dianthus caryophyllus L.).

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. 'Francesco' was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568,887,315 bp, consisting of 45,088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16,644 bp and 60,737 bp, respectively, and the longest scaffold was 1,287,144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼ 98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp.