Pattern analysis suggests that phosphoenolpyruvate carboxylase in maturing soybean seeds promotes the accumulation of protein.

The protein and oil contents in soybean seeds are major factors in seed quality. Seed proteins and oils are synthesized from sucrose and nitrogenous compounds transported into maturing seeds. In this study, we compared changes in the activity of phosphoenolpyruvate carboxylase (PEPC) and the accumulation profiles of protein and oil in maturing seeds of two soybean cultivars, which exhibit different protein and oil contents in seeds, to determine the interrelationships of them. A principal component analysis indicated a concordance of seed PEPC activity with the protein content, but did not with the oil content. PEPC activity per seed was highest in the late maturation stage, when the physiological status of the vegetative organs drastically changed. The high-protein cultivar had higher PEPC activity compared to the low-protein cultivar. These results highlight the biological role of PEPC in the synthesis of protein, therefore it was implied that PEPC could be a biomarker in soybean breeding.Abbreviations: ANOVA: analysis of variance; DS: developmental stage; DW: dry weight; FW: fresh weight; NIR: near infrared; PEP(C): phosphoenolpyruvate (carboxylase); PC(A): principal component (analysis); S.E.: standard error; WC: water content.

Randomized, Double-Blind, Crossover Clinical Trial of the Effect of Calcium Alginate in Noodles on Postprandial Blood Glucose Level.

We conducted a prospective, randomized, double-blind, 3-group, 3-phase crossover study to evaluate the effect of calcium alginate (Ca-Alg) on the postprandial increase of blood glucose in 15 healthy adult subjects who were given udon noodles containing or not containing Ca-Alg (5 or 8%). The value of ΔCmax (difference between the maximum (Cmax) and pre-feeding (C0) blood glucose levels) was significantly reduced in both Ca-Alg groups, and the area under the blood glucose level-time curve over 120 min (ΔAUC, with C0 as the baseline) was also significantly reduced. Thus, supplementation of noodles with Ca-Alg significantly suppressed both the peak postprandial blood glucose level and the total amount of glucose absorption. Blood calcium (Ca) concentration was significantly increased at 120 min after ingestion, but there was no marked change of other parameter values. A questionnaire indicated that addition of Ca-Alg did not affect the acceptability of the noodles. These results indicate that Ca-Alg might a useful food additive for helping to prevent lifestyle-related diseases without adversely affecting individual eating habits.

CATchUP: A Web Database for Spatiotemporally Regulated Genes.

For proper control of biological activity, some key genes are highly expressed in a particular spatiotemporal domain. Mining of such spatiotemporally expressed genes using large-scale gene expression data derived from a broad range of experimental sources facilitates our understanding of genome-scale functional gene networks. However, comprehensive information on spatiotemporally expressed genes is lacking in plants. To collect such information, we devised a new index, Δdmax, which is the maximum difference in relative gene expression levels between sample runs which are neighboring when sorted by the levels. Employing this index, we comprehensively evaluated transcripts using large-scale RNA sequencing (RNA-Seq) data stored in the Sequence Read Archive for eight plant species: Arabidopsis thaliana (Arabidopsis), Solanum lycopersicum (tomato), Solanum tuberosum (potato), Oryza sativa (rice), Sorghum bicolor (sorghum), Vitis vinifera (grape), Medicago truncatula (Medicago), and Glycine max (soybean). Based on the frequency distribution of the Δdmax values, approximately 70,000 transcripts showing 0.3 or larger Δdmax values were extracted for the eight species. Information on these genes including the Δdmax values, functional annotations, conservation among species, and experimental conditions where the genes show high expression levels is provided in a new database, CATchUP (http://plantomics.mind.meiji.ac.jp/CATchUP). The CATchUP database assists in identifying genes specifically expressed under particular conditions with powerful search functions and an intuitive graphical user interface.

Comparative analysis of microRNA profiles of rice anthers between cool-sensitive and cool-tolerant cultivars under cool-temperature stress.

Plants subjected to abiotic stress can regulate gene expression post-transcriptionally by means of small RNAs such as microRNAs. Cool-temperature stress causes abnormal tapetum hypertrophy in rice anthers, leading to pollen sterility. As a first step toward understanding the molecular mechanisms of cool tolerance in developing anthers of rice, we report here a comprehensive comparative analysis of microRNAs between cool-sensitive Sasanishiki and cool-tolerant Hitomebore cultivars. High-throughput Illumina sequencing revealed 241 known and 46 novel microRNAs. Interestingly, 15 of these microRNAs accumulated differentially in the two cultivars at the uninucleate microspore stage under cool conditions. Inverse correlations between expression patterns of microRNAs and their target genes were confirmed by quantitative RT-PCR analysis, and cleavage sites of some of the target genes were determined by 5' RNA ligase-mediated RACE experiments. Thus, our data are useful resources to elucidate microRNA-mediated mechanism(s) of cool tolerance in rice anthers at the booting stage.

Recommended Daily Dose of Sesame Lignans Has No Influence on Oral Absorption of P-Glycoprotein Substrates in Rats.

Sesamin (SM) and episesamin (ESM) are constituents of sesame seeds, which are used in health foods and considered to have various beneficial effects in the prevention of lifestyle-related diseases. P-Glycoprotein (P-gp) is an ATP-binding cassette transporter involved in drug absorption in the human gastrointestinal tract. A recent report indicated that SM influences P-gp-mediated drug transport. In the present study, we investigated whether SM and ESM inhibit P-gp in vitro, using Caco-2 cells and the typical P-gp substrates rhodamine123 (Rho123) and fexofenadine. SM and ESM showed no effect on accumulation of these compounds, indicating that SM and ESM do not influence P-gp function. In addition, an in vivo study using Rho123 indicated that SM and ESM do not affect absorption of P-gp substrates. Overall, these results suggest that health foods containing SM and ESM are unlikely to interact with P-gp substrates.

Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing.

Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen-stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms.

Comprehensive network analysis of anther-expressed genes in rice by the combination of 33 laser microdissection and 143 spatiotemporal microarrays.

Co-expression networks systematically constructed from large-scale transcriptome data reflect the interactions and functions of genes with similar expression patterns and are a powerful tool for the comprehensive understanding of biological events and mining of novel genes. In Arabidopsis (a model dicot plant), high-resolution co-expression networks have been constructed from very large microarray datasets and these are publicly available as online information resources. However, the available transcriptome data of rice (a model monocot plant) have been limited so far, making it difficult for rice researchers to achieve reliable co-expression analysis. In this study, we performed co-expression network analysis by using combined 44 K agilent microarray datasets of rice, which consisted of 33 laser microdissection (LM)-microarray datasets of anthers, and 143 spatiotemporal transcriptome datasets deposited in RicexPro. The entire data of the rice co-expression network, which was generated from the 176 microarray datasets by the Pearson correlation coefficient (PCC) method with the mutual rank (MR)-based cut-off, contained 24,258 genes and 60,441 genes pairs. Using these datasets, we constructed high-resolution co-expression subnetworks of two specific biological events in the anther, "meiosis" and "pollen wall synthesis". The meiosis network contained many known or putative meiotic genes, including genes related to meiosis initiation and recombination. In the pollen wall synthesis network, several candidate genes involved in the sporopollenin biosynthesis pathway were efficiently identified. Hence, these two subnetworks are important demonstrations of the efficiency of co-expression network analysis in rice. Our co-expression analysis included the separated transcriptomes of pollen and tapetum cells in the anther, which are able to provide precise information on transcriptional regulation during male gametophyte development in rice. The co-expression network data presented here is a useful resource for rice researchers to elucidate important and complex biological events.

Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices.

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5h following LPS treatment (100microg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10microM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices.

Morphological and gene expression analysis under cool temperature conditions in rice anther development.

Cool temperature conditions are known to lead to pollen sterility in rice. Pollen sterility is an agriculturally important phenomenon because it imparts a large influence directly on rice yield. However, cool temperature stress tolerance varies among rice cultivars and avoidance of cool temperature stress is difficult by practical method of agriculture. In this study using two rice cultivars, Hitomebore (high tolerance) and Sasanishiki (low tolerance), we analyzed morphological features and gene expression profiles, under cool temperature stress, in anther development of rice. Hitomebore was given cool temperature stress (19 degrees C) at flowering stage, and showed 87.3% seed fertility. Meanwhile, the seed fertility decreased to 41.7% in the case of Sasanishiki. A transverse section of Hitomebore anther revealed that the degradation of the tapetum started at the uninucleate microspore stage, and the tapetum had completely vanished at mature stage. The tapetum provides nutrients for pollen development, and its degradation occurs at a late stage in pollen development. In contrast, degradation of the tapetum did not occur at the uninucleate microspore stage in Sasanishiki, and the tapetum was clearly intact at mature stage, suggesting that tapetum degradation is critical for accurate pollen development and cool temperature tolerance correlated with the degree of tapetum degeneration. In gene expression analysis of anther, 356 genes that showed different expression levels between two cultivars at cool temperatures were found. These genes will lead to understanding the mechanism of cool temperature stress response in rice pollen development and the identification of genes involved in accurate tapetum degradation.

The effect of dimerumic acid on LPS-induced downregulation of Mrp2 in the rat.

Oxidative stress is known to be a common feature of cholestatic syndrome. Lipopolysaccharide (LPS) induces cholestasis, causing multidrug resistance-associated protein 2 (Mrp2) downregulation in two different ways: early retrieval from the canalicular membrane and the latter event of reduced mRNA expression. However, the triggering factor for LPS-induced cholestasis is not fully understood. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and traditional Chinese medicine, on endotoxin-induced Mrp2 downregulation in rat liver. At 3h following LPS injection (4mg/kg body weight), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of tight junctions in the liver. Pretreatment with DMA (12mg/kg body weight) counteracted LPS-induced subcellular distribution, and decreased the bile flow rate and biliary glutathione (GSH) excretion. At 12h following LPS injection, Mrp2 protein and mRNA expression were significantly decreased by 58% and 7%, respectively. In contrast, pretreatment with DMA did not have any effect on the decreased Mrp2 expression and biliary excretion of GSH induced by LPS exposure. Taken together, our data clearly indicate that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress, while the long-term transcriptional regulation of Mrp2 expression did not depend on the intracellular redox status.

Cytoplasmic-nuclear genomic barriers in rice pollen development revealed by comparison of global gene expression profiles among five independent cytoplasmic male sterile lines.

Cytoplasmic male sterility (CMS) is one of the most ideal phenomena known in higher plants to describe the incompatibilities between mitochondrial-nuclear genomic interactions. To elucidate the dependency of pollen development on mitochondrial genotypes and cytoplasmic-nuclear genomic barriers, we employed five CMS isogenic lines of rice, CW-, W11-, LD-, BT- and WA-type CMS lines, that exhibit distinct pollen-defective phenotypes, and we characterized the CMS phenotypes and the nuclear gene expression patterns in conjunction with their mitochondrial genomic structures. These five CMS lines carried independent mitotypes, and W11, LD and BT mitochondrial genomes were relatively close with respect to their phylogeny. In anthers at the uninucleate microspore and bicellular pollen stages, 8,199 genes significantly changed their expression in at least one of the CMS lines. Common expression patterns were observed in BT, LD and W11 after k-means clustering. Among the genes encoding putative mitochondrial proteins, ALTERNATIVE OXIDASE 1A, a gene for the well-known mitochondrial stress marker, was included in the group ectopically up-regulated in anthers at the bicellular pollen stage of BT, LD and W11. Several other clusters were also regulated in a cytoplasm-specific manner during pollen development. These clear similarities in gene regulatory networks of BT-, LD- and W11-CMS lines indicate that the phylogenetic relationships of the mitochondrial genotypes are strongly correlated with nuclear gene expression patterns and pollen abortion phenotypes, providing evidence of the mitochondrial epistacy over the nuclear genome during pollen development.

High temperatures cause male sterility in rice plants with transcriptional alterations during pollen development.

Plant male reproductive development is highly organized and sensitive to various environmental stressors, including high temperature. We have established an experimental procedure to evaluate high temperature injury in japonica rice plants. High temperature treatment (39 degrees C/30 degrees C) starting at the microspore stage repeatedly reduced spikelet fertility in our system. Morphological observations revealed that pollen viability in plants exposed to high temperatures was lower than that in control plants. Most pollen grains in high temperature-treated plants displayed a normal round shape and stained reddish purple with Alexander's reagent; however, the pollen grains were very poorly attached and displayed limited germination on the stigma. To investigate gene regulatory mechanisms in the anther in high temperature environments, DNA microarray analysis was performed by comparing non-treated samples with samples treated with 2-4 d of high heat. Genes responsive to high temperatures were identified from clustering of microarray data. Among these, at least 13 were designated as high temperature-repressed genes in the anther. Expression analyses revealed that these genes were expressed specifically in the immature anther mainly in the tapetum at the microspore stage and down-regulated after 1 d of high temperature. The expression levels of Osc6, OsRAFTIN and TDR, which are tapetum-specific genes, were unaffected by high temperatures. These results suggest that not all tapetal genes are inhibited by increased temperatures and the tapetum itself is not degraded in such an environment. However, high temperatures may disrupt some of the tapetum functions required for pollen adhesion and germination on the stigma.

Various spatiotemporal expression profiles of anther-expressed genes in rice.

The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.

Separated transcriptomes of male gametophyte and tapetum in rice: validity of a laser microdissection (LM) microarray.

In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum.