Unified Interpretations of Two Kinds of Needle-Shaped Precipitates Using Transmission Electron Microscopy and Small-Angle Neutron Scattering in Aged Al-Mg2Si(-Cu) Alloys.

This study investigates the nanostructural properties of pseudo-binary Al-1.0Mg2Si (mass%) alloys with and without 0.5Cu using transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). The TEM results show that both alloys exhibit extra electron diffraction spots related to MgSiMg second clusters at peak-aged conditions. High-resolution TEM images have revealed that the second cluster exists as a needle-shaped precipitate that is shorter and thicker than the ß″ phase. We found that the second cluster, which we referred to as the R phase in this paper, is more likely to form partially along the longitudinal axis of a random-type precipitate. Thus, the atomic arrangement in the random-type precipitate is not completely random. SANS is used to quantify the size and volume fraction of the observed needle-shaped precipitates since the R phase is difficult to observe with TEM. The R phase forms even in the Cu-free alloy, but the volume fraction is low, and the growth and formation are retarded near the peak-aged conditions. Undoubtedly, the Cu addition has the effect of stabilizing the growth of the R phase and also promoting its formation. Therefore, the R phase also contributes to the increase in hardness at both under- and peak-aged conditions in the Cu-containing alloy in addition to the strengthening ß″ phases.

Horizontally Transferred DNA in the Genome of the Fungus Pyricularia oryzae is Associated With Repressive Histone Modifications.

Horizontal gene transfer (HGT) is a means of exchanging genetic material asexually. The process by which horizontally transferred genes are domesticated by the host genome is of great interest but is not well understood. In this study, we determined the telomere-to-telomere genome sequence of the wheat-infecting Pyricularia oryzae strain Br48. SNP analysis indicated that the Br48 strain is a hybrid of wheat- and Brachiaria-infecting strains by a sexual or parasexual cross. Comparative genomic analysis identified several megabase-scale "insertions" in the Br48 genome, some of which were possibly gained by HGT-related events from related species, such as P. pennisetigena or P. grisea. Notably, the mega-insertions often contained genes whose phylogeny is not congruent with the species phylogeny. Moreover, some of the genes have a close homolog even in distantly related organisms, such as basidiomycetes or prokaryotes, implying the involvement of multiple HGT events. Interestingly, the levels of the silent epigenetic marks H3K9me3 and H3K27me3 in a genomic region tended to be negatively correlated with the phylogenetic concordance of genes in the same region, suggesting that horizontally transferred DNA is preferentially targeted for epigenetic silencing. Indeed, the putative HGT-derived genes were activated when MoKmt6, the gene responsible for H3K27me3 modification, was deleted. Notably, these genes also tended to be up-regulated during infection, suggesting that they are now under host control and have contributed to establishing a fungal niche. In conclusion, this study suggests that epigenetic modifications have played an important role in the domestication of HGT-derived genes in the P. oryzae genome.

Characterization of the Functions of Starch Synthase IIIb Expressed in the Vegetative Organs of Rice (Oryza sativa L.).

Rice is the model C3 crop for investigating the starch biosynthesis mechanism in endosperm because of its importance in grain production. However, little is known about starch biosynthesis in the vegetative organs of rice. In this study, we used novel rice mutants by inserting Tos17 into the starch synthase (SS) IIIb gene, which is mainly expressed in the leaf sheath (LS) and leaf blade (LB), and an ss1 mutant to clarify the differences in roles among SS isozymes during starch biosynthesis. Native polyacrylamide gel electrophoresis (PAGE)/activity staining for SS, using LS and LB of ss mutants, revealed that the lowest migrating SS activity bands on the gel were derived from SSIIIb activity and those of two ss3b mutants were not detected. The apparent amylose content of LS starch of ss3b mutants increased. Moreover, the chain-length distribution and size-exclusion chromatography analysis using ss mutants showed that SSIIIb and SSI synthesize the B2-B3 chain and A-B1 chain of amylopectin in the LS and LB respectively. Interestingly, we also found that starch contents were decreased in the LS and LB of ss3b mutants, although SSI deficiency did not affect the starch levels. All these results indicated that SSIIIb synthesizes the long chain of amylopectin in the LS and LB similar to SSIIIa in the endosperm, while SSI synthesizes the short chain in the vegetative organ as the same in the endosperm.

Electron tomography imaging methods with diffraction contrast for materials research.

Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) enable the visualization of three-dimensional (3D) microstructures ranging from atomic to micrometer scales using 3D reconstruction techniques based on computed tomography algorithms. This 3D microscopy method is called electron tomography (ET) and has been utilized in the fields of materials science and engineering for more than two decades. Although atomic resolution is one of the current topics in ET research, the development and deployment of intermediate-resolution (non-atomic-resolution) ET imaging methods have garnered considerable attention from researchers. This research trend is probably not irrelevant due to the fact that the spatial resolution and functionality of 3D imaging methods of scanning electron microscopy (SEM) and X-ray microscopy have come to overlap with those of ET. In other words, there may be multiple ways to carry out 3D visualization using different microscopy methods for nanometer-scale objects in materials. From the above standpoint, this review paper aims to (i) describe the current status and issues of intermediate-resolution ET with regard to enhancing the effectiveness of TEM/STEM imaging and (ii) discuss promising applications of state-of-the-art intermediate-resolution ET for materials research with a particular focus on diffraction contrast ET for crystalline microstructures (superlattice domains and dislocations) including a demonstration of in situ dislocation tomography.

CO2-Responsive CCT Protein Stimulates the Ectopic Expression of Particular Starch Biosynthesis-Related Enzymes, Which Markedly Change the Structure of Starch in the Leaf Sheaths of Rice.

CO2-responsive CCT protein (CRCT) is suggested to be a positive regulator of starch biosynthesis in the leaf sheaths of rice, regulating the expression levels of starch biosynthesis-related genes. In this study, the effects of CRCT expression levels on the expression of starch biosynthesis-related enzymes and the quality of starch were studied. Using native-PAGE/activity staining and immunoblotting, we found that the protein levels of starch synthase I, branching enzyme I, branching enzyme IIa, isoamylase 1 and phosphorylase 1 were largely correlated with the CRCT expression levels in the leaf sheaths of CRCT transgenic lines. In contrast, the CRCT expression levels largely did not affect the expression levels and/or activities of starch biosynthesis-related enzymes in the leaf blades and endosperm tissues. The analysis of the chain-length distribution of starch in the leaf sheaths showed that short chains with a degree of polymerization from 5 to 14 were increased in the overexpression lines but decreased in the knockdown lines. The amylose content of starch in the leaf sheath was greatly increased in the overexpression lines. In contrast, the molecular weight of the amylopectin of starch in the leaf sheath of overexpression lines did not change compared with those of the non-transgenic rice. These results suggest that CRCT can control the quality and the quantity of starch in the leaf sheath by regulating the expression of particular starch biosynthesis-related enzymes.

A fungal Argonaute interferes with RNA interference.

Small RNA (sRNA)-mediated gene silencing phenomena, exemplified by RNA interference (RNAi), require a unique class of proteins called Argonautes (AGOs). An AGO protein typically forms a protein-sRNA complex that contributes to gene silencing using the loaded sRNA as a specificity determinant. Here, we show that MoAGO2, one of the three AGO genes in the fungus Pyricularia oryzae (Magnaporthe oryzae) interferes with RNAi. Gene knockout (KO) studies revealed that MoAGO1 and MoAGO3 additively or redundantly played roles in hairpin RNA- and retrotransposon (MAGGY)-triggered RNAi while, surprisingly, the KO mutants of MoAGO2 (Δmoago2) showed elevated levels of gene silencing. Consistently, transcript levels of MAGGY and mycoviruses were drastically reduced in Δmoago2, supporting the idea that MoAGO2 impeded RNAi against the parasitic elements. Deep sequencing analysis revealed that repeat- and mycovirus-derived small interfering RNAs were mainly associated with MoAGO2 and MoAGO3, and their populations were very similar based on their size distribution patterns and positional base preference. Site-directed mutagenesis studies indicated that sRNA binding but not slicer activity of MoAGO2 was essential for the ability to diminish the efficacy of RNAi. Overall, these results suggest a possible interplay between distinct sRNA-mediated gene regulation pathways through a competition for sRNA.

Essential role of the PSI-LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions.

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m-2 s-1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI-LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.

Starch Content in Leaf Sheath Controlled by CO2-Responsive CCT Protein is a Potential Determinant of Photosynthetic Capacity in Rice.

CO2-responsive CCT protein (CRCT) is the suggested positive regulator of starch synthesis in vegetative organs, particularly the leaf sheath of rice. In this study, we analyzed the effects of the starch level in the leaf sheath on the photosynthetic rate in the leaf blade using CRCT overexpression and RNA interference (RNAi) knockdown transgenic rice grown under ambient (38 Pa) or elevated (100 Pa) CO2 conditions. In leaf sheath, the starch content was markedly changed in relation to CRCT expression levels under both CO2 conditions. In contrast, the soluble sugar and starch contents of the leaf blade were markedly increased in the knockdown line grown under elevated CO2 conditions. The overexpression or RNAi knockdown of CRCT did not cause large effects on the photosynthetic rate of the transgenic lines grown under ambient CO2 condition. However, the photosynthetic rate of the overexpression line was enhanced, while that of the knockdown line was substantially decreased under elevated CO2 conditions. These photosynthetic rates were weakly correlated with the nitrogen contents and negatively correlated with the total non-structural carbohydrate contents. Thus, the capacity for starch synthesis in leaf sheath, which is controlled by CRCT, can indirectly affect the carbohydrate content, and then the photosynthetic rate in the leaf blade of rice grown under elevated CO2 conditions.

Suppression of Chloroplastic Alkenal/One Oxidoreductase Represses the Carbon Catabolic Pathway in Arabidopsis Leaves during Night.

Lipid-derived reactive carbonyl species (RCS) possess electrophilic moieties and cause oxidative stress by reacting with cellular components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the detoxification of lipid-derived RCS, especially α,ß-unsaturated carbonyls. In this study, we aimed to evaluate the physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA), and RNA interference knockdown, aor (RNAi), lines. We found that both aor mutants showed smaller plant sizes than wild-type plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wild-type and aor (RNAi) plants showed similar CO2 assimilation rates; however, the respiration rate was lower in aor (RNAi) than in wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation during the night was suppressed in aor (RNAi). In contrast, the phenotype of aor (RNAi) was rescued when aor (RNAi) plants were grown under constant light conditions. These results indicate that the smaller plant sizes observed in aor mutants grown under day/night cycle conditions were attributable to the decrease in carbon utilization during the night. Here, we propose that the detoxification of lipid-derived RCS by AtAOR in chloroplasts contributes to the protection of dark respiration and supports plant growth during the night.

Hardness and microstructural variation of Al-Mg-Mn-Sc-Zr alloy.

Variations of Vickers hardness were observed in Al-Mg-Mn alloy and Al-Mg-Mn-Sc-Zr alloy at different ageing times, ranging from a peak value of 81.2 HV at 54 ks down to 67.4 HV at 360 ks, below the initial hardness value, 71.8 HV at 0 ks for the case of Al-Mg-Mn-Sc-Zr alloy. Microstructures of samples at each ageing stage were examined carefully by transmission electron microscopes (TEMs) both in two-dimensions and three-dimensions. The presence of different types, densities, and sizes of particles were observed dispersed spherical Al3Sc1-xZrx and also block-shaped Al3Sc precipitates growing along <100>Al with facets {100} and {110} of the precipitates. TEM analysis both in two-dimensions and three-dimensions, performed on various samples, confirmed the direct correlation between the hardness and the density of Al3Sc.

MoSET1 (Histone H3K4 Methyltransferase in Magnaporthe oryzae) Regulates Global Gene Expression during Infection-Related Morphogenesis.

Here we report the genetic analyses of histone lysine methyltransferase (KMT) genes in the phytopathogenic fungus Magnaporthe oryzae. Eight putative M. oryzae KMT genes were targeted for gene disruption by homologous recombination. Phenotypic assays revealed that the eight KMTs were involved in various infection processes at varying degrees. Moset1 disruptants (Δmoset1) impaired in histone H3 lysine 4 methylation (H3K4me) showed the most severe defects in infection-related morphogenesis, including conidiation and appressorium formation. Consequently, Δmoset1 lost pathogenicity on wheat host plants, thus indicating that H3K4me is an important epigenetic mark for infection-related gene expression in M. oryzae. Interestingly, appressorium formation was greatly restored in the Δmoset1 mutants by exogenous addition of cAMP or of the cutin monomer, 16-hydroxypalmitic acid. The Δmoset1 mutants were still infectious on the super-susceptible barley cultivar Nigrate. These results suggested that MoSET1 plays roles in various aspects of infection, including signal perception and overcoming host-specific resistance. However, since Δmoset1 was also impaired in vegetative growth, the impact of MoSET1 on gene regulation was not infection specific. ChIP-seq analysis of H3K4 di- and tri-methylation (H3K4me2/me3) and MoSET1 protein during infection-related morphogenesis, together with RNA-seq analysis of the Δmoset1 mutant, led to the following conclusions: 1) Approximately 5% of M. oryzae genes showed significant changes in H3K4-me2 or -me3 abundance during infection-related morphogenesis. 2) In general, H3K4-me2 and -me3 abundance was positively associated with active transcription. 3) Lack of MoSET1 methyltransferase, however, resulted in up-regulation of a significant portion of the M. oryzae genes in the vegetative mycelia (1,491 genes), and during infection-related morphogenesis (1,385 genes), indicating that MoSET1 has a role in gene repression either directly or more likely indirectly. 4) Among the 4,077 differentially expressed genes (DEGs) between mycelia and germination tubes, 1,201 and 882 genes were up- and down-regulated, respectively, in a Moset1-dependent manner. 5) The Moset1-dependent DEGs were enriched in several gene categories such as signal transduction, transport, RNA processing, and translation.

Inhibition of abscission layer formation by an interaction of two seed-shattering loci, sh4 and qSH3, in rice.

Loss of seed shattering was one of the key phenotypic changes selected for in the domestication of many crop species. Asian cultivated rice, Oryza sativa L., was domesticated from its wild ancestor, O. rufipogon, and three seed-shattering loci, qSH1, sh4 and qSH3, have been reported to be involved in the loss of seed shattering in cultivated rice. Here, we analysed the seed-shattering behaviour of wild rice using introgression lines carrying the cultivated alleles from O. sativa Nipponbare in the genetic background of wild rice, O. rufipogon W630. We first carried out fine mapping of the qSH3 region and found that the qSH3 locus is localized in an 850-kb region on chromosome 3. We then analysed the effects of the Nipponbare alleles at sh4 and qSH3 on seed-shattering behaviour in wild rice, as a mutation at qSH1 was not commonly found in rice cultivars. Seed-shattering behaviour did not change in the two types of introgression line independently carrying the Nipponbare-homozygous alleles at sh4 or qSH3 in the genetic background of wild rice. However, the introgression lines having the Nipponbare-homozygous alleles at both sh4 and qSH3 showed a reduction in the degree of seed shattering. Histological and scanning electron microscopy analyses revealed that abscission layer formation was inhibited around the vascular bundles in these lines. Since the qSH3 region, as well as sh4, has been shown to be under artificial selection, the interaction of mutations at these two loci may have played a role in the initial loss of seed shattering during rice domestication.

Regulation of photochemical energy transfer accompanied by structural changes in thylakoid membranes of heat-stressed wheat.

Photosystems of higher plants alleviate heat-induced damage in the presence of light under moderate stressed conditions; however, in the absence of light (i.e., in the dark), the same plants are damaged more easily. (Yamauchi and Kimura, 2011) We demonstrate that regulating photochemical energy transfer in heat-treated wheat at 40 °C with light contributed to heat tolerance of the photosystem. Chlorophyll fluorescence analysis using heat-stressed wheat seedlings in light showed increased non-photochemical quenching (NPQ) of chlorophyll fluorescence, which was due to thermal dissipation that was increased by state 1 to state 2 transition. Transmission electron microscopy revealed structural changes in thylakoid membranes, including unstacking of grana regions under heat stress in light. It was accompanied by the phosphorylation of thylakoid proteins such as D1 and D2 proteins and the light harvesting complex II proteins Lhcb1 and Lhcb2. These results suggest that heat stress at 40 °C in light induces state 1 to state 2 transition for the preferential excitation of photosystem I (PSI) by phosphorylating thylakoid proteins more strongly. Structural changes of thylakoid membrane also assist the remodeling of photosystems and regulation of energy distribution by transition toward state 2 probably contributes to plastoquione oxidation; thus, light-driven electrons flowing through PSI play a protective role against PSII damage under heat stress.

Is the fungus Magnaporthe losing DNA methylation?

The long terminal repeat retrotransposon, Magnaporthe gypsy-like element (MAGGY), has been shown to be targeted for cytosine methylation in a subset of Magnaporthe oryzae field isolates. Analysis of the F1 progeny from a genetic cross between methylation-proficient (Br48) and methylation-deficient (GFSI1-7-2) isolates revealed that methylation of the MAGGY element was governed by a single dominant gene. Positional cloning followed by gene disruption and complementation experiments revealed that the responsible gene was the DNA methyltransferase, MoDMT1, an ortholog of Neurospora crassa Dim-2. A survey of MAGGY methylation in 60 Magnaporthe field isolates revealed that 42 isolates from rice, common millet, wheat, finger millet, and buffelgrass were methylation proficient while 18 isolates from foxtail millet, green bristlegrass, Japanese panicgrass, torpedo grass, Guinea grass, and crabgrass were methylation deficient. Phenotypic analyses showed that MoDMT1 plays no major role in development and pathogenicity of the fungus. Quantitative polymerase chain reaction analysis showed that the average copy number of genomic MAGGY elements was not significantly different between methylation-deficient and -proficient field isolates even though the levels of MAGGY transcript were generally higher in the former group. MoDMT1 gene sequences in the methylation-deficient isolates suggested that at least three independent mutations were responsible for the loss of MoDMT1 function. Overall, our data suggest that MoDMT1 is not essential for the natural life cycle of the fungus and raise the possibility that the genus Magnaporthe may be losing the mechanism of DNA methylation on the evolutionary time scale.

Social disorganization/social fragmentation and risk of depression among older people in Japan: multilevel investigation of indices of social distance.

Previous studies reported that social disorganization/fragmentation could predict mental well-being of residents in a community. The aim of this study is to examine how area and individual level of social distance could predict likelihood of mental health among older people in Japan. We empirically derived an index of "social distance" by taking averaged differences in sociodemographic characteristics that are income, education, hometown of origin, the duration of residency, and life stage, between the study participants and their neighbors. We used the study participants (n = 9147) from the Aichi Gerontological Evaluation Study, which targeted residents with aged 65 years or over in a central part in Japan. Depressive symptoms of the study participants were assessed using the short version of the Geriatric Depression Scale (GDS-15). We also tested if area-level social capital would moderate the association between social distance and depressive symptoms. Using multilevel analyses, we found that higher social distance from neighbors was associated with increased depressive symptoms, independently of respondents' own values of income and educational attainment. At the individual level, each standard deviation in income-based and education-based social distance was associated with an odds ratio for depressive symptoms of 1.15 (95% CI: 1.01-1.30) and 1.17 (95% CI: 1.03-1.32), respectively. However, the area-aggregated indices of social distance were not associated with depressive symptoms. Additionally, area-level social capital indicating higher levels of trust between neighbors and social participation, buffered the adverse effect of social distance on depressive risk. In an instance of the "dark side" of social capital, we also found that stronger social cohesion increased depressive symptoms for residents whose hometown of origin differed from the communities where they currently resided.

Neighborhood social capital and crime victimization: comparison of spatial regression analysis and hierarchical regression analysis.

Crime is an important determinant of public health outcomes, including quality of life, mental well-being, and health behavior. A body of research has documented the association between community social capital and crime victimization. The association between social capital and crime victimization has been examined at multiple levels of spatial aggregation, ranging from entire countries, to states, metropolitan areas, counties, and neighborhoods. In multilevel analysis, the spatial boundaries at level 2 are most often drawn from administrative boundaries (e.g., Census tracts in the U.S.). One problem with adopting administrative definitions of neighborhoods is that it ignores spatial spillover. We conducted a study of social capital and crime victimization in one ward of Tokyo city, using a spatial Durbin model with an inverse-distance weighting matrix that assigned each respondent a unique level of "exposure" to social capital based on all other residents' perceptions. The study is based on a postal questionnaire sent to 20-69 years old residents of Arakawa Ward, Tokyo. The response rate was 43.7%. We examined the contextual influence of generalized trust, perceptions of reciprocity, two types of social network variables, as well as two principal components of social capital (constructed from the above four variables). Our outcome measure was self-reported crime victimization in the last five years. In the spatial Durbin model, we found that neighborhood generalized trust, reciprocity, supportive networks and two principal components of social capital were each inversely associated with crime victimization. By contrast, a multilevel regression performed with the same data (using administrative neighborhood boundaries) found generally null associations between neighborhood social capital and crime. Spatial regression methods may be more appropriate for investigating the contextual influence of social capital in homogeneous cultural settings such as Japan.

Step-templated CVD growth of aligned graphene nanoribbons supported by a single-layer graphene film.

We present chemical vapor deposition (CVD) growth of a hybrid structure of aligned graphene nanoribbons (GNRs) supported by a single-layer graphene sheet. The step structure created on the epitaxial Co film is used to segregate arrays of aligned GNRs. Reflecting the highly ordered step structure of the Co catalyst, straight nanoribbons with high aspect ratio (>100) are formed. Analysis suggests that a large-area, single-layer graphene film also grows over the aligned GNRs, making a GNR-graphene hybrid structure. We also demonstrate the isolation of aligned GNRs by oxygen plasma treatment or partial transfer of the hybrid film. These findings on the formation of highly aligned GNRs give new insights into the formation mechanism of graphene and can be applied for more advanced graphene structure for future electronics.

Enhanced effects of nonisotopic hafnium chloride in methanol as a substitute for uranyl acetate in TEM contrast of ultrastructure of fungal and plant cells.

This ultrastructural study showed that nonisotopic methanolic hafnium chloride and aqueous lead solution was an excellent new electron stain for enhancing TEM contrasts of fungal and plant cell structures. The ultrastructural definition provided by the new stain was often superior to that provided by conventional staining with uranyl acetate and lead. Definition of fine ultrastructure was also supported by quantitative data on TEM contrast ratios of organelles and components in fungal and plant cells. In particular, polysaccharides, which were localized in cell walls, glycogen particles, starch grains, and plant Golgi vesicle components, were much more reactive to the new stain than to the conventional one. The new nonisotopic stain is useful for enhancing the contrast of ultrastructure in biological tissues and is a safer alternative to uranyl acetate.