Exploring the diversity of galls on Artemisia indica induced by Rhopalomyia species through morphological and transcriptome analyses.

Plant galls generated by insects have highly organized structures, providing nutrients and shelter to the insects living within them. Most research on the physiological and molecular mechanisms of gall development has focused on single galls. To understand the diversity of gall development, we examined five galls with different morphologies generated by distinct species of Rhopalomyia (gall midge; Diptera: Cecidomyiidae) on a single host plant of Artemisia indica var. maximowiczii (Asteraceae). Vasculature developed de novo within the galls, indicating active transport of nutrients between galls and the host plant. Each gall had a different pattern of vasculature and lignification, probably due to differences in the site of gall generation and the gall midge species. Transcriptome analysis indicated that photosynthetic and cell wall-related genes were down-regulated in leaf and stem galls, respectively, compared with control leaf and stem tissues, whereas genes involved in floral organ development were up-regulated in all types of galls, indicating that transformation from source to sink organs occurs during gall development. Our results help to understand the diversity of galls on a single herbaceous host plant.

Relationships of the Surface Charge of Low-Density Lipoprotein (LDL) with the Serum LDL-Cholesterol and Atherosclerosis Levels in a Japanese Population: The DOSANCO Health Study.

AIM: This study investigated the associations of the surface charge of low-density lipoprotein (LDL) with the serum LDL-cholesterol and atherosclerosis levels in a community-based Japanese population. METHODS: The study had a cross-sectional design and included 409 community residents aged 35-79 years who did not take medications for dyslipidemia. The potential electric charge of LDL and the zeta potential, which indicate the surface charge of LDL, were measured by laser Doppler microelectrophoresis. The correlations of the zeta potential of LDL (-mV) with the serum LDL-cholesterol levels (mg/dL), cardio-ankle vascular index (CAVI), and serum high-sensitivity C-reactive protein (hsCRP) levels (log-transformed values, mg/L) were examined using Pearson's correlation coefficient (r). Linear regression models were constructed to examine these associations after adjusting for potential confounding factors. RESULTS: A total of 201 subjects with correctly stored samples were included in the primary analysis for zeta potential measurement. An inverse correlation was observed between the LDL zeta potential and the serum LDL-cholesterol levels (r=-0.20; p=0.004). This inverse association was observed after adjusting for sex, age, dietary cholesterol intake, smoking status, alcohol intake, body mass index, and the serum levels of the major classes of free fatty acids (standardized ß=-6.94; p=0.005). However, the zeta potential of LDL showed almost no association with CAVI or the serum hsCRP levels. Similar patterns were observed in the 208 subjects with compromised samples as well as all the original 409 subjects. CONCLUSION: A higher electronegative surface charge of LDL was associated with lower serum LDL-cholesterol levels in the general Japanese population.

Binding properties of recombinant LDL receptor and LOX-1 receptor to LDL measured using bio-layer interferometry and atomic force microscopy.

Oxidation of low-density lipoproteins (LDLs) triggers a recognition by scavenger receptors such as lectin-like oxidized LDL receptor-1 (LOX-1) and is related to inflammation and cardiovascular diseases. Although LDLs that are recognized by LOX-1 can be risk-related LDLs, conventional LDL detection methods using commercially available recombinant receptors remain undeveloped. Using a bio-layer interferometry (BLI), we investigated the binding of recombinant LOX-1 (reLOX-1) and LDL receptors to the oxidized LDLs. The recombinant LDL receptor preferably bound minimally modified LDLs, while the reLOX-1 recognized extensively oxidized LDLs. An inversed response of the BLI was observed during the binding in the case of reLOX-1. AFM study showed that the extensively oxidized LDLs and aggregates of LDLs were observed on the surface, supporting the results. Altogether, a combined use of these recombinant receptors and the BLI method is useful in detecting high-risk LDLs such as oxidized LDLs and modified LDLs.

Histone deacetylation regulates de novo shoot regeneration.

During de novo plant organ regeneration, auxin induction mediates the formation of a pluripotent cell mass called callus, which regenerates shoots upon cytokinin induction. However, molecular mechanisms underlying transdifferentiation remain unknown. Here, we showed that the loss of HDA19, a histone deacetylase (HDAC) family gene, suppresses shoot regeneration. Treatment with an HDAC inhibitor revealed that the activity of this gene is essential for shoot regeneration. Further, we identified target genes whose expression was regulated through HDA19-mediated histone deacetylation during shoot induction and found that ENHANCER OF SHOOT REGENERATION 1 and CUP-SHAPED COTYLEDON 2 play important roles in shoot apical meristem formation. Histones at the loci of these genes were hyperacetylated and markedly upregulated in hda19. Transient ESR1 or CUC2 overexpression impaired shoot regeneration, as observed in hda19. Therefore, HDA19 mediates direct histone deacetylation of CUC2 and ESR1 loci to prevent their overexpression at the early stages of shoot regeneration.

Ab-GALFA, A bioassay for insect gall formation using the model plant Arabidopsis thaliana.

Insect galls are abnormal plant organs formed by gall-inducing insects to provide shelter and nutrients for themselves. Although insect galls are spatialized complex structures with unique shapes and functions, the molecular mechanism of the gall formation and the screening system for the gall inducing effectors remains unknown. Here, we demonstrate that an extract of a gall-inducing aphid, Schlechtendalia chinensis, induces an abnormal structure in the root-tip region of Arabidopsis seedlings. The abnormal structure is composed of stem-like cells, vascular, and protective tissues, as observed in typical insect galls. Furthermore, we confirm similarities in the gene expression profiles between the aphid-treated seedlings and the early developmental stages of Rhus javanica galls formed by S. chinensis. Based on the results, we propose a model system for analyzing the molecular mechanisms of gall formation: the Arabidopsis-based Gall-Forming Assay (Ab-GALFA). Ab-GALFA could be used not only as a model to elucidate the mechanisms underlying gall formation, but also as a bioassay system to isolate insect effector molecules of gall-induction.

Gene-flow investigation between garden and wild roses planted in close distance.

Rose is a major ornamental plant, and a lot of cultivars with attractive morphology, color and scent have been generated by classical breeding. Recent progress of genetic modification produces a novel cultivar with attractive features. In both cases, a major problem is the gene-flow from cultivated or genetically modified (GM) plants to wild species, causing reduction of natural population. To investigate whether gene-flow occurs in wild species, molecular analysis with DNA markers with higher efficient technique is useful. Here we investigated the gene-flow from cultivated roses (Rosa×hybrida) to wild rose species planted in close distance in the field. The overlapping flowering periods and visiting insects suggest that pollens were transported by insects between wild and cultivated roses. We examined the germination ratio of seeds from wild species, and extracted DNA and checked with KSN and APETALA2 (AP2) DNA markers to detect transposon insertions. Using two markers, we successfully detected the outcross between wild and cultivated roses. For higher efficiency, we established a bulking method, where DNA, leaves or embryos were pooled, enabling us to that check the outcross of many plants. Our results suggest that wild species and garden cultivars can cross in close distance, so that they should be planted in distance, and checked the outcross with multiple DNA markers.

Evaluating the effectiveness of a geostatistical approach with groundwater flow modeling for three-dimensional estimation of a contaminant plume.

When assessing the risk from an underground environment that is contaminated by radioactive nuclides and hazardous chemicals and planning for remediation, the contaminant plume distribution and the associated uncertainty from measured data should be estimated accurately. While the release history of the contaminant plume may be unknown, the extent of the plume caused by a known source and the associated uncertainty can be calculated inversely from the concentration data using a geostatistical method that accounts for the temporal correlation of its release history and groundwater flow modeling. However, the preceding geostatistical approaches have three drawbacks: (1) no applications of the three-dimensional plume estimation using concentration data from multiple depths in real situations, (2) no constraints for the estimation of the plume distribution, which can yield negative concentration and large uncertainties, and (3) few applications to actual cases with multiple contaminants. To address these problems, the non-negativity constraint using Gibbs sampling was incorporated into the geostatistical method with groundwater flow modeling for contaminant plume estimation. This method was then tested on groundwater contamination in the Gloucester landfill in Ontario, Canada, using three-dimensional contaminant transport model and concentration data from multiple depths. The method was applied to three water soluble organic contaminants: 1,4-dioxane, tetrahydrofuran, and diethyl ether. The effectiveness of the proposed method was verified by the general agreement of the calculated plume distributions of the three contaminants with concentration data from 66 points in 1982 (linear correlation coefficient of about 0.7). In particular, the reproduced peak of 1,4-dioxane corresponding to the large disposal in 1978 was more accurate than the result of preceding minimum relative entropy-based studies. The same peak also appeared in the tetrahydrofuran and diethyl ether distributions approximately within the range of the retardation factor derived from the fraction of organic carbon.

Non-cell-autonomous regulation of petal initiation in Arabidopsis thaliana.

In many flowering plants, petals initiate in alternate positions from first whorl sepals, suggesting possible signaling between sepal boundaries and petal initiation sites. PETAL LOSS (PTL) and RABBIT EARS (RBE) regulate petal initiation in Arabidopsis thaliana and their transcripts are expressed in sepal boundary and petal initiation sites, respectively, suggesting that PTL acts in a non-cell-autonomous manner. Here, we determined that cells expressing PTL and RBE fusion proteins did not overlap but were adjacent, confirming the non-cell-autonomous function of PTL. Genetic ablation of intersepal cells by expressing the diphtheria toxin-A chain gene driven by the PTL promoter resulted in flowers lacking petals, suggesting these cells are required for petal initiation. Transcriptome analysis combined with a PTL induction system revealed 42 genes that were upregulated under PTL activation, including UNUSUAL FLORAL ORGANS (UFO), which likely plays an important role in petal initiation. These findings suggest a molecular mechanism in which PTL indirectly regulates petal initiation and UFO mediates positional signaling between the sepal boundary and petal initiation sites.

Histone chaperone-mediated co-expression assembly of tetrasomes and nucleosomes.

The nucleosome, a basic unit of chromatin found in all eukaryotes, is thought to be assembled through the orchestrated activity of several histone chaperones and chromatin assembly factors in a stepwise manner, proceeding from tetrasome assembly, to H2A/H2B deposition, and finally to formation of the mature nucleosome. In this study, we demonstrate chaperone-mediated assembly of both tetrasomes and nucleosomes on the well-defined Widom 601 positioning sequence using a co-expression/reconstitution wheat germ cell-free system. The purified tetrasomes and nucleosomes were positioned around the center of a given sequence. The heights and diameters were measured by atomic force microscopy. Together with the reported unmodified native histones produced by the wheat germ cell-free platform, our method is expected to be useful for downstream applications in the field of chromatin research.

Recent Progress Regarding the Molecular Aspects of Insect Gall Formation.

Galls are characteristic plant structures formed by cell size enlargement and/or cell proliferation induced by parasitic or pathogenic organisms. Insects are a major inducer of galls, and insect galls can occur on plant leaves, stems, floral buds, flowers, fruits, or roots. Many of these exhibit unique shapes, providing shelter and nutrients to insects. To form unique gall structures, gall-inducing insects are believed to secrete certain effector molecules and hijack host developmental programs. However, the molecular mechanisms of insect gall induction and development remain largely unknown due to the difficulties associated with the study of non-model plants in the wild. Recent advances in next-generation sequencing have allowed us to determine the biological processes in non-model organisms, including gall-inducing insects and their host plants. In this review, we first summarize the adaptive significance of galls for insects and plants. Thereafter, we summarize recent progress regarding the molecular aspects of insect gall formation.

Reduction in organ-organ friction is critical for corolla elongation in morning glory.

In complex structures such as flowers, organ-organ interactions are critical for morphogenesis. The corolla plays a central role in attracting pollinators: thus, its proper development is important in nature, agriculture, and horticulture. Although the intraorgan mechanism of corolla development has been studied, the importance of organ-organ interactions during development remains unknown. Here, using corolla mutants of morning glory described approximately 200 years ago, we show that glandular secretory trichomes (GSTs) regulate floral organ interactions needed for corolla morphogenesis. Defects in GST development in perianth organs result in folding of the corolla tube, and release of mechanical stress by sepal removal restores corolla elongation. Computational modeling shows that the folding occurs because of buckling caused by mechanical stress from friction at the distal side of the corolla. Our results suggest a novel function of GSTs in regulating the physical interaction of floral organs for macroscopic morphogenesis of the corolla.

Dose Estimation of Landfill Disposal of Removed Soil Generated Outside Fukushima Prefecture.

ABSTRACT: Dose estimation was conducted by assuming landfill disposal of removed soil generated outside the Fukushima Prefecture by each local town and in a lump sum. Because the radioactivity of removed soil is lower than that of specified waste that was generated at Fukushima Prefecture and the radioactivity concentration is 100,000 Bq kg-1 or less, simple landfill covered with 30 cm of non-contaminated soil was used. The exposure doses of loading/unloading, transportation, and landfill workers and the public residing near the repository site were estimated. Furthermore, migration of cesium into groundwater because of precipitation and using the contaminated groundwater for drinking and agricultural water was evaluated, and exposure doses regarding farmers and the ingestion of agricultural products were estimated. It was confirmed that estimated exposure doses during landfill were less than 1 mSv y-1, and those for after landfill were 0.01 mSv y-1.

Establishment of the Embryonic Shoot Meristem Involves Activation of Two Classes of Genes with Opposing Functions for Meristem Activities.

The shoot meristem, a stem-cell-containing tissue initiated during plant embryogenesis, is responsible for continuous shoot organ production in postembryonic development. Although key regulatory factors including KNOX genes are responsible for stem cell maintenance in the shoot meristem, how the onset of such factors is regulated during embryogenesis is elusive. Here, we present evidence that the two KNOX genes STM and KNAT6 together with the two other regulatory genes BLR and LAS are functionally important downstream genes of CUC1 and CUC2, which are a redundant pair of genes that specify the embryonic shoot organ boundary. Combined expression of STM with any of KNAT6, BLR, and LAS can efficiently rescue the defects of shoot meristem formation and/or separation of cotyledons in cuc1cuc2 double mutants. In addition, CUC1 and CUC2 are also required for the activation of KLU, a cytochrome P450-encoding gene known to restrict organ production, and KLU counteracts STM in the promotion of meristem activity, providing a possible balancing mechanism for shoot meristem maintenance. Together, these results establish the roles for CUC1 and CUC2 in coordinating the activation of two classes of genes with opposite effects on shoot meristem activity.

Development of an automated two pronuclei detection system on time-lapse embryo images using deep learning techniques.

PURPOSE: To establish an automated pronuclei determination system by analysis using deep learning technology which is able to effectively learn with limited amount of supervised data. METHODS: An algorithm was developed by explicitly incorporating human observation where the outline around pronuclei is being observed in determining the number of pronuclei. Supervised data were selected from the time-lapse images of 300 pronuclear stage embryos per class (total 900 embryos) clearly classified by embryologists as 0PN, 1PN, and 2PN. One-hundred embryos per class (a total of 300 embryos) were used for verification data. The verification data were evaluated for the performance of detection in the number of pronuclei by regarding the results consistent with the judgment of the embryologists as correct answers. RESULTS: The sensitivity rates of 0PN, 1PN, and 2PN were 99%, 82%, and 99%, respectively, and the overlapping 2PN being difficult to determine by microscopic observation alone could also be appropriately assessed. CONCLUSIONS: This study enabled the establishment of the automated pronuclei determination system with the precision almost equivalent to highly skilled embryologists.

Investigation on the Epoxidation of Piperitenone, and Structure-activity Relationships of Piperitenone Oxide for Differentiation-inducing Activity.

Piperitenone oxide, a major chemical constituent of the essential oil of spearmint, Mentha spicata, induces differentiation in human colon cancer RCM-1 cells. In this study, piperitenone oxide and trans-piperitenone dioxide were prepared as racemic forms by epoxidation of piperitenone. The relative configuration between two epoxides in piperitenone dioxide was determined to be trans by 1H NMR analysis and nuclear Overhauser effect spectroscopy (NOESY) in conjunction with density functional theory (DFT) calculations. Optical resolution of (±)-piperitenone oxide by high-performance liquid chromatography (HPLC) using a chiral stationary phase (CSP) afforded both enantiomers with over 98% enantiomeric excess (ee). Evaluation of the differentiation-inducing activity of the synthetic compounds revealed that the epoxide at C-1 and C-6 in piperitenone oxide is important for the activity, and (+)-piperitenone oxide has stronger activity than (-)-piperitenone oxide. The results obtained in this study provide new information on the application of piperitenone oxide and spearmint for differentiation-inducing therapy. Furthermore, natural piperitenone oxide was isolated from M. spicata. The enantiomeric excess of the isolated natural piperitenone oxide was 66% ee. Epoxidation of piperitenone with hydrogen peroxide proceeded in a phosphate buffer under weak basic conditions to give (±)-piperitenone oxide. These results suggest that the nonenzymatic epoxidation of piperitenone, which causes a decrease in the enantiomeric excess of natural piperitenone oxide, is accompanied by an enzymatic epoxidation in the biosynthesis of piperitenone oxide.

Reprogramming of the Developmental Program of Rhus javanica During Initial Stage of Gall Induction by Schlechtendalia chinensis.

Insect galls are unique organs that provide shelter and nutrients to the gall-inducing insects. Although insect galls are fascinating structures for their unique shapes and functions, the process by which gall-inducing insects induce such complex structures is not well understood. Here, we performed RNA-sequencing-based comparative transcriptomic analysis of the early developmental stage of horned gall to elucidate the early gall-inducing process carried out by the aphid, Schlechtendalia chinensis, in the Chinese sumac, Rhus javanica. There was no clear similarity in the global gene expression profiles between the gall tissue and other tissues, and the expression profiles of various biological categories such as phytohormone metabolism and signaling, stress-response pathways, secondary metabolic pathways, photosynthetic reaction, and floral organ development were dramatically altered. Particularly, master transcription factors that regulate meristem, flower, and fruit development, and biotic and abiotic stress-responsive genes were highly upregulated, whereas the expression of genes related to photosynthesis strongly decreased in the early stage of the gall development. In addition, we found that the expression of class-1 KNOX genes, whose ectopic overexpression is known to lead to the formation of de novo meristematic structures in leaf, was increased in the early development stage of gall tissue. These results strengthen the hypothesis that gall-inducing insects convert source tissues into fruit-like sink tissues by regulating the gene expression of host plants and demonstrate that such manipulation begins from the initial process of gall induction.

DOSE ESTIMATION FOR CONTAMINATED SOIL STORAGE IN LIVING ENVIRONMENT.

After the Fukushima Nuclear Power Plant accident, most of radiocesium-contaminated soil generated from decontamination activities outside Fukushima prefecture has been stored at decontamination sites such as schools, parks and residential lands (storage at sites) according to the Decontamination Guidelines. However, additional exposure due to the present storage has not been evaluated. Moreover, entering storage sites, which is not restricted for storage at sites, was not considered in safety assessment conducted in the guidelines. To continue the storage and confirm the effectiveness, understanding of present possible exposures is important. In this study, we evaluated exposure doses for residents and users of storage sites based on the present situation. As a result, annual doses due to residence were 10-2 to 10-3 mSv y-1 and doses due to annual entries were of the order of 10-3 mSv y-1. Hence, we confirmed that the exposure due to present storage outside Fukushima is significantly <1 mSv y-1.

Morphological and Genetic Diversities of Habenaria radiata (Orchidaceae) in the Kinki Area, Japan.

Floral organs have evolved from leaves for reproduction, and the morphological analyses help to understand the plant diversity and evolution. Habenaria radiata (syn. Pecteilis radiata) is a terrestrial orchid living in wetlands in Japan, Russia, South Korea, and China. The habitats of this plant in Japan have been reduced because of environmental destruction and overexploitation, and thus it is on the Red List of Japan as a Near Threatened species. One of the three petals of the H. radiata flower is called a lip or labellum, which resembles a flying white bird, egret, or white heron, with its proposed function being to attract pollinators. To understand the diversity of H. radiata plants in different areas, we examined the lip morphology and phylogeny of populations from eight habitats in the Kinki area, Japan. The complex shapes of the lips were quantified and presented as a radar chart, enabling characterization of the morphological difference among populations. Phylogenetic analysis with microsatellite markers that we generated showed the variation of genetic diversity among populations, suggesting the different degrees of inbreeding, outbreeding, and vegetative propagation. Our approach offers a basic method to characterize the morphological and genetic diversity in natural populations.

Substantial Narrowing on the Width of "Concentration Window" of Hydrothermal ZnO Nanowires via Ammonia Addition.

A crystal growth of hydrothermal ZnO nanowires essentially requires a concentration control within so-called "concentration window", where the anisotropic crystal growth of ZnO nanowires preferentially occurs. Although understanding what exactly determines the width of "concentration window" is important to tailor the anisotropic crystal growth process, the fundamental knowledge as to "concentration window" is still scarce. Here we report the effect of ammonia addition on the width of "concentration window" using conventional hydrothermal ZnO nanowire growth. We found that the ammonia addition substantially narrows the width of "concentration window". Within the narrow range of zinc complex concentration, we found a significant increase of growth rate (up to 2000 nm/h) of ZnO nanowires. The narrowed "concentration window" and the resultant increased growth rate by the ammonia addition can be understood in terms of synchronized effects of both (1) a reduction of zinc hydroxide complex (precursor) concentration and (2) a fast rate limiting process of ligand exchange between different zinc complexes. Thus, the present knowldege as to "concentration window" will accelerate further tailoring an anisotropic crystal growth of hydrothermal ZnO nanowires.

Comparative transcriptome analysis of galls from four different host plants suggests the molecular mechanism of gall development.

Galls are plant structures generated by gall-inducing organisms including insects, nematodes, fungi, bacteria and viruses. Those made by insects generally consist of inner callus-like cells surrounded by lignified hard cells, supplying both nutrients and protection to the gall insects living inside. This indicates that gall insects hijack developmental processes in host plants to generate tissues for their own use. Although galls are morphologically diverse, the molecular mechanism for their development remains poorly understood. To identify genes involved in gall development, we performed RNA-sequencing based transcriptome analysis for leaf galls. We examined the young and mature galls of Glochidion obovatum (Phyllanthaceae), induced by the micromoth Caloptilia cecidophora (Lepidoptera: Gracillariidae), the leaf gall from Eurya japonica (Pentaphylacaceae) induced by Borboryctis euryae (Lepidoptera: Gracillariidae), and the strawberry-shaped leaf gall from Artemisia montana (Asteraceae) induced by gall midge Rhopalomyia yomogicola (Oligotrophini: Cecidomyiidae). Gene ontology (GO) analyses suggested that genes related to developmental processes are up-regulated, whereas ones related to photosynthesis are down-regulated in these three galls. Comparison of transcripts in these three galls together with the gall on leaves of Rhus javanica (Anacardiaceae), induced by the aphid Schlechtendalia chinensis (Hemiptera: Aphidoidea), suggested 38 genes commonly up-regulated in galls from different plant species. GO analysis showed that peptide biosynthesis and metabolism are commonly involved in the four different galls. Our results suggest that gall development involves common processes across gall inducers and plant taxa, providing an initial step towards understanding how they manipulate host plant developmental systems.