Wheat Cybrid Plants, OryzaWheat, Regenerated from Wheat-Rice Hybrid Zygotes via in Vitro Fertilization System Possess Wheat-Rice Hybrid Mitochondria.

Hybridization generates biodiversity, and wide hybridization plays a pivotal role in enhancing and broadening the useful attributes of crops. The hybridization barrier between wheat and rice, the two most important cereals, was recently overcome by in vitro production of allopolyploid wheat-rice hybrid zygotes, which can develop and grow into mature plants. In the study, genomic sequences and compositions of the possible hybrid plants were investigated through short- and long-read sequencing analyses and fluorescence in situ hybridization (FISH)-based visualization. The possible hybrid possessed whole wheat nuclear and cytoplasmic DNAs and rice mitochondrial (mt) DNA, along with variable retention rates of rice mtDNA ranging from 11% to 47%. The rice mtDNA retained in the wheat cybrid, termed Oryzawheat, can be transmitted across generations. In addition to mitochondrial hybridization, translocation of rice chromosome 1 into wheat chromosome 6A was detected in a F1 hybrid individual. OryzaWheat can provide a new horizon for utilizing inter-subfamily genetic resources among wheat and rice belonging to different subfamilies, Pooideae and Ehrhartoideae, respectively.

Temporal changes in transcripts of miniature inverted-repeat transposable elements during rice endosperm development.

The repression of transcription from transposable elements (TEs) by DNA methylation is necessary to maintain genome integrity and prevent harmful mutations. However, under certain circumstances, TEs may escape from the host defense system and reactivate their transcription. In Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), DNA demethylases target the sequences derived from TEs in the central cell, the progenitor cell for the endosperm in the female gametophyte. Genome-wide DNA demethylation is also observed in the endosperm after fertilization. In the present study, we used a custom microarray to survey the transcripts generated from TEs during rice endosperm development and at selected time points in the embryo as a control. The expression patterns of TE transcripts are dynamically up- and downregulated during endosperm development, especially those of miniature inverted-repeat TEs (MITEs). Some TE transcripts were directionally controlled, whereas the other DNA transposons and retrotransposons were not. We also discovered the NUCLEAR FACTOR Y binding motif, CCAAT, in the region near the 5' terminal inverted repeat of Youren, one of the transcribed MITEs in the endosperm. Our results uncover dynamic changes in TE activity during endosperm development in rice.

DOMINANT AWN INHIBITOR Encodes the ALOG Protein Originating from Gene Duplication and Inhibits AWN Elongation by Suppressing Cell Proliferation and Elongation in Sorghum.

The awn, a needle-like structure extending from the tip of the lemma in grass species, plays a role in environmental adaptation and fitness. In some crops, awns appear to have been eliminated during domestication. Although numerous genes involved in awn development have been identified, several dominant genes that eliminate awns are also known to exist. For example, in sorghum (Sorghum bicolor), the dominant awn-inhibiting gene has been known since 1921; however, its molecular features remain uncharacterized. In this study, we conducted quantitative trait locus analysis and a genome-wide association study of awn-related traits in sorghum and identified DOMINANT AWN INHIBITOR (DAI), which encodes the ALOG family protein on chromosome 3. DAI appeared to be present in most awnless sorghum cultivars, likely because of its effectiveness. Detailed analysis of the ALOG protein family in cereals revealed that DAI originated from a duplication of its twin paralog (DAIori) on chromosome 10. Observations of immature awns in near-isogenic lines revealed that DAI inhibits awn elongation by suppressing both cell proliferation and elongation. We also found that only DAI gained a novel function to inhibit awn elongation through an awn-specific expression pattern distinct from that of DAIori. Interestingly, heterologous expression of DAI with its own promoter in rice inhibited awn elongation in the awned cultivar Kasalath. We found that DAI originated from gene duplication, providing an interesting example of gain-of-function that occurs only in sorghum but shares its functionality with rice and sorghum.

Phylogenetic distribution and expression pattern analyses identified a divergent basal body assembly protein involved in land plant spermatogenesis.

Land plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is a continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning spermatogenesis remain to be elucidated. We successfully identified candidate genes involved in spermatogenesis, deeply divergent BLD10s, by computational analyses combining multiple methods and omics data. We then examined the functions of BLD10s in the liverwort Marchantia polymorpha and the moss Physcomitrium patens. MpBLD10 and PpBLD10 are required for normal basal body (BB) and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, and thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. We identified orthologs of MpBLD10 and PpBLD10 in diverse Streptophyta and found that MpBLD10 and PpBLD10 are orthologous to BLD10/CEP135 family proteins, which function in BB assembly. However, BLD10s evolved especially quickly in land plants and MpBLD10 might have acquired additional functions in spermatozoid formation through rapid molecular evolution.

Expression and localization of tight junction-related proteins in adult rat pituitary stem/progenitor cell niches.

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100ß, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.

Repression of Nitrogen Starvation Responses by Members of the Arabidopsis GARP-Type Transcription Factor NIGT1/HRS1 Subfamily.

Nitrogen (N) is often a limiting nutrient whose availability determines plant growth and productivity. Because its availability is often low and/or not uniform over time and space in nature, plants respond to variations in N availability by altering uptake and recycling mechanisms, but the molecular mechanisms underlying how these responses are regulated are poorly understood. Here, we show that a group of GARP G2-like transcription factors, Arabidopsis thaliana NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 proteins (NIGT1/HRS1s), are factors that bind to the promoter of the N starvation marker NRT2.4 and repress an array of N starvation-responsive genes under conditions of high N availability. Transient assays and expression analysis demonstrated that NIGT1/HRS1s are transcriptional repressors whose expression is regulated by N availability. We identified target genes of the NIGT1/HRS1s by genome-wide transcriptome analyses and found that they are significantly enriched in N starvation response-related genes, including N acquisition, recycling, remobilization, and signaling genes. Loss of NIGT1/HRS1s resulted in deregulation of N acquisition and accumulation. We propose that NIGT1/HRS1s are major regulators of N starvation responses that play an important role in optimizing N acquisition and utilization under fluctuating N conditions.

Laser Vaporization of Intracoronary Thrombus and Identifying Plaque Morphology in ST-Segment Elevation Myocardial Infarction as Assessed by Optical Coherence Tomography.

OBJECTIVES: We evaluated the thrombus-vaporizing effect of excimer laser coronary angioplasty (ELCA) in patients with ST-segment elevation myocardial infarction (STEMI) by optical coherence tomography (OCT). BACKGROUND: Larger intracoronary thrombus elevates the risk of interventional treatment and mortality in patients with STEMI. METHODS: A total of 92 patients with STEMI who presented within 24 hours from the onset and underwent ELCA following manual aspiration thrombectomy (MT) were analyzed. RESULTS: The mean baseline thrombolysis in myocardial infarction flow grade was 0.4 ± 0.6, which subsequently improved to 2.3 ± 0.7 after MT (p < 0.0001) and 2.7 ± 0.5 after ELCA (p=0.0001). The median residual thrombus volume after MT was 65.7 mm3, which significantly reduced to 47.5 mm3 after ELCA (p < 0.0001). Plaque rupture was identified by OCT in only 22 cases (23.9%) after MT, but was distinguishable in 36 additional cases after ELCA (total: 58 cases; 63.0%). Ruptured lesions contained a higher proportion of red thrombus than nonruptured lesions (75.9% vs. 43.3%, p=0.001). Significantly larger thrombus burden after MT (69.6 mm3 vs. 56.3 mm3, p < 0.05) and greater thrombus reduction by ELCA (21.2 mm3 vs. 11.8 mm3, p < 0.01) were observed in ruptured lesions than nonruptured lesions. CONCLUSIONS: ELCA effectively vaporized intracoronary thrombus in patients with STEMI even after MT. Lesions with plaque rupture contained larger thrombus burden that was frequently characterized by red thrombus and more effectively reduced by ELCA.

Rapid Increase of Gastrointestinal P-Glycoprotein Functional Activity in Response to Etoposide Stimulation.

We previously reported that exposure of human colon adenocarcinoma (Caco-2) cells to the bitter substance phenylthiocarbamide (PTC) rapidly enhanced the transport function of P-glycoprotein (P-gp). In this study, we investigated the short-term effect of etoposide, another bitter-tasting P-gp substrate, on P-gp transport function in the same cell line. We found that etoposide exposure significantly increased both the P-gp protein level in the plasma membrane fraction and the efflux rate of rhodamine123 (Rho123) in Caco-2 cells within 10 min. The efflux ratio (ratio of the apparent permeability coefficient in the basal-to-apical direction to that in the apical-to-basal direction) of Rho123 in etoposide-treated cells was also significantly increased compared with the control. These results indicated that etoposide rapidly enhances P-gp function in Caco-2 cells. In contrast, P-gp expression in whole cells at both the mRNA and protein level was unchanged by etoposide exposure, compared with the levels in non-treated cells. Furthermore, etoposide increased the level of phosphorylated ezrin, radixin and moesin (P-ERM) proteins in the plasma membrane fraction of Caco-2 cells within 10 min. P-gp functional changes were blocked by YM022, an inhibitor of cholecystokinin (CCK) receptor. These results suggest that etoposide induces release of CCK, causing activation of the CCK receptor followed by phosphorylation of ERM proteins, which recruit intracellular P-gp for trafficking to the gastrointestinal membrane, thereby increasing the functional activity of P-gp.

Functional Alterations of Multidrug Resistance-Associated Proteins 2 and 5, and Breast Cancer Resistance Protein upon Snail-Induced Epithelial-Mesenchymal Transition in HCC827 Cells.

Our previous report indicated that Snail-induced epithelial-mesenchymal transition (EMT) enhanced P-glycoprotein (P-gp) function and drug resistance to P-gp substrate anticancer drug in a human non-small cell lung cancer (NSCLC) cell line, HCC827. Our objective is to evaluate the changes in the mRNA and protein expression levels and the functions of multidrug resistance-associated protein (MRP) 2, MRP5 and breast cancer resistance protein (BCRP). Snail-expressing HCC827 cells showed increased mRNA levels of Snail and a mesenchymal marker vimentin, and decreased mRNA levels of an epithelial marker E-cadherin after transduction, indicating that Snail had induced EMT consistent with our previous reports. The mRNA level of MRP2 was significantly decreased, while that of MRP5 remained unchanged, in Snail-expressing cells. The expression levels of MRP2 and MRP5 proteins in whole-cell homogenate were unchanged in Snail-expressing cells, but MRP5 protein showed significantly increased membrane localization. Snail-transduction increased the efflux transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDCF), a substrate of MRP2, 3 and 5. This increase was blocked by MK571, which inhibits MRP1, 2, and 5. Toxicity of cisplatin, a substrate of MRP2 and 5, was significantly decreased in Snail-expressing cells. BCRP mRNA and protein levels were both decreased in Snail-expressing cells, which showed an increase in the intracellular accumulation of 7-ethyl-10-hydroxycamptothecin (SN-38), a BCRP substrate, resulting in reduced viability. These results suggested that MRP5 function appears to be increased via an increase in membrane localization, whereas the BCRP function is decreased via a decrease in the expression level in HCC827 cells with Snail-induced EMT.

RAD-seq-Based High-Density Linkage Map Construction and QTL Mapping of Biomass-Related Traits in Sorghum using the Japanese Landrace Takakibi NOG.

Sorghum [Sorghum bicolor (L.) Moench] grown locally by Japanese farmers is generically termed Takakibi, although its genetic diversity compared with geographically distant varieties or even within Takakibi lines remains unclear. To explore the genomic diversity and genetic traits controlling biomass and other physiological traits in Takakibi, we focused on a landrace, NOG, in this study. Admixture analysis of 460 sorghum accessions revealed that NOG belonged to the subgroup that represented Asian sorghums, and it was only distantly related to American/African accessions including BTx623. In an attempt to dissect major traits related to biomass, we generated a recombinant inbred line (RIL) from a cross between BTx623 and NOG, and we constructed a high-density linkage map based on 3,710 single-nucleotide polymorphisms obtained by restriction-site-associated DNA sequencing of 213 RIL individuals. Consequently, 13 fine quantitative trait loci (QTLs) were detected on chromosomes 2, 3, 6, 7, 8 and 9, which included five QTLs for days to heading, three for plant height (PH) and total shoot fresh weight and two for Brix. Furthermore, we identified two dominant loci for PH as being identical to the previously reported dw1 and dw3. Together, these results corroborate the diversified genome of Japanese Takakibi, while the RIL population and high-density linkage map generated in this study will be useful for dissecting other important traits in sorghum.

The plant-type phosphoenolpyruvate carboxylase Gmppc2 is developmentally induced in immature soy seeds at the late maturation stage: a potential protein biomarker for seed chemical composition.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon-fixing enzyme with critical roles in seed development. Previously we observed a positive correlation between PEPC activity and protein content in mature seeds among soybean cultivars and varietal differences of PEPC activity in immature seeds, which is concordant with seed protein accumulation. Here, we report a PEPC isoform (Gmppc2) which is preferentially expressed in immature soybean seeds at the late maturation stage. Gmppc2 was co-expressed with enzyme genes involved in starch degradation: α-amylase, hexokinase, and α-glucan phosphorylase. Gmppc2 was developmentally induced in the external seed coats, internal seed coats, hypocotyls, and cotyledons at the late maturation stage. The expression of Gmppc2 protein was negatively regulated by the application of a nitrogen fertilizer, which suppressed nodule formation. These results imply that Gmppc2 is involved in the metabolism of nitrogen originated from nodules into seeds, and Gmppc2 might be applicable as a biomarker of seed protein content.Abbreviations: PEP: phosphoenolpyruvate; PEPC: phosphoenolpyruvate carboxylase; RNA-Seq: RNA sequencing; PCA: principal component analysis; SE: standard error.

Effect of salt tolerance on biomass production in a large population of sorghum accessions.

Salinity causes major reductions in cultivated land area, crop productivity, and crop quality, and salt-tolerant crops have been required to sustain agriculture in salinized areas. The annual C4 crop plant Sorghum bicolor (L.) Moench is salt tolerant, with large variation among accessions. Sorghum's salt tolerance is often evaluated during early growth, but such evaluations are weakly related to overall performance. Here, we evaluated salt tolerance of 415 sorghum accessions grown in saline soil (0, 50, 100, and 150 mM NaCl) for 3 months. Some accessions produced up to 400 g per plant of biomass and showed no growth inhibition at 50 mM NaCl. Our analysis indicated that the genetic factors that affected biomass production under 100 mM salt stress were more different from those without salt stress, comparing to the differences between those under 50 mM and 100 mM salt stress. A genome-wide association study for salt tolerance identified two single-nucleotide polymorphisms (SNPs) that were significantly associated with biomass production, only at 50 mM NaCl. Additionally, two SNPs were significantly associated with salt tolerance index as an indicator for growth response of each accession to salt stress. Our results offer candidate genetic resources and SNP markers for breeding salt-tolerant sorghum.

Expression of Genes from Paternal Alleles in Rice Zygotes and Involvement of OsASGR-BBML1 in Initiation of Zygotic Development.

Upon fertilization in angiosperms, one sperm cell fuses with the egg cell to produce a zygote, and, via karyogamy, the parental genetic information is combined to form the diploid zygotic genome. Recently, analyses with parentally imbalanced rice zygotes indicated that parental genomes are utilized synergistically in zygotes with different functions, and that genes transcribed from the paternal or maternal allele might play important roles in zygotic development. Herein, we first conducted single nucleotide polymorphism-based mRNA-sequencing using intersubspecific rice zygotes. Twenty-three genes, with paternal allele-specific expression in zygotes, were identified, and, surprisingly, their allele dependencies in the globular-like embryo tended to be biallelic. This suggests that the paternal-dependent expression of these genes is temporary, occurring during the early stages of zygote development. Of the 23 genes, we focused on Oryza sativa Apospory-specific Genome Region (ASGR)-BABY-BOOM LIKE (BBML) 1 (OsASGR-BBML1), presumed to encode an AP2-transcription factor, due to its reported role in zygotic development. Interestingly, ectopic expression of OsASGR-BBML1 in egg cells induced nuclear and cell divisions, indicating that exogenously expressed OsASGR-BBML1 converts the proliferation status of the egg cell from quiescent to active. In addition, the suppression of the function of OsASGR-BBML1 and its homologs in zygotes resulted in the developmental arrest, suggesting that OsASGR-BBML1 possesses an important role in initiating zygotic development. Monoallelic or preferential gene expression from the paternal genome in the zygote might be a safety mechanism allowing egg cells to suppress the gene expression cascade toward early embryogenesis that is normally triggered by fusion with a sperm cell.

Testosterone and androstenedione are endogenous substrates of P-glycoprotein.

Raised brain levels of testosterone (Tes), as well as single nucleotide polymorphisms of P-glycoprotein (P-gp) that cause impaired transport function, are associated with increased risk of suicide. Here, we examined whether Tes and its precursors and metabolites are substrates of P-gp, using several in vitro methods. In ATPase assay, increased ATP consumption was observed as the concentrations of Tes, dihydroepiandrosterone (Dhea), androstenedione (Ado), and dihydrotestosterone (Dht), but not androstenediol (Adol), were increased, suggesting that these four androgens are transported by P-gp. Furthermore, Tes and Ado, though not Dhea or Dht, increased the intracellular accumulation of Rhodamine 123 (Rho123), a typical substrate of P-gp, in a P-gp-overexpressing cell line, suggesting that they inhibit Rho123 efflux and thus are substrates or inhibitors of P-gp. A membrane permeability study using P-gp-overexpressing cells in Transwell inserts indicated that the permeability coefficients of both Ado and Tes in the basal-to-apical direction (excretion) are significantly higher than those in the apical-to-basal direction. Moreover, transport of both Ado and Tes was significantly suppressed by verapamil, a typical P-gp inhibitor. These results indicate that Tes and Ado are endogenous substrates of P-gp. These findings provide a physiological basis for understanding previously reported associations of P-gp dysfunction and raised brain levels of Tes with suicidal behavior, and may open up new possibilities for treating patients at risk of suicide.

Reduction Effect of Calcium Alginate on Blood Triglyceride Levels Causing the Inhibition of Hepatic and Total Body Accumulation of Fat in Rats.

In this study, rats were fed a high-fat diet containing calcium alginate (Ca-Alg) for 5 weeks to examine the effects of Ca-Alg on lipid metabolism including triglyceride (TG) levels in the blood. We also investigated the mechanism of the TG-reducing effect of Alg in vitro. Rats were randomized into 5 groups: high-fat diet group (14% (w/w) lard, HF); three Ca-Alg-containing diet groups (2.5, 5 or 10% (w/w) Ca-Alg) and a resistant maltodextrin (RMD) diet group as a positive control (with 5% (w/w) RMD). The 10% Ca-Alg group showed a significant reduction of body weight increase from the 7th day. In addition, the increase of TG in blood was significantly suppressed, and the amount of TG excreted in feces was increased. Increase of body fat mass was in the order HF > RMD > Ca-Alg 2.5% > Ca-Alg 5% > Ca-Alg 10%, while the total weight of the extracted fat tissues was significantly reduced in the RMD, 5% and 10% Ca-Alg groups. Hepatic pathology showed clear circular vacuoles apparently representing TG accumulation in the HF group, while fewer vacuoles were seen in the Ca-Alg groups. The results of in vitro experiments indicated that Ca-Alg does not directly inhibit lipase activity, but may suppress absorption of TG by forming non-absorbable macromolecular micelles containing TG. These results suggest that Ca-Alg promotes excretion and suppresses absorption of TG, leading to reduced blood TG levels, and decreased hepatic and total body accumulation of fat. The findings should be helpful for designing future clinical trials.

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.

Efficacy of Cutting Balloon Angioplasty for Chronic Total Occlusion of Femoropopliteal Arteries.

BACKGROUND: Chronic total occlusion (CTO) of femoropopliteal artery (FP) continues to be a lesion subset where maintaining long-term patency after endovascular treatment is challenging. We evaluated the efficacy of cutting balloon angioplasty (CBA) for de novo FP-CTOs in patients with symptomatic lower limb ischemia. METHODS: Seventy-three limbs of 67 symptomatic patients with de novo FP-CTOs successfully recanalized using CBA alone were enrolled in this study. Primary patency was defined as the absence of recurrent symptoms and no deterioration of the ankle-brachial index (ABI) >0.10 from the immediate postinterventional value. RESULTS: The mean age was 73.5 ± 7.3 years, and 59.7% of patients had diabetes mellitus. Most lesions were classified as Trans-Atlantic Inter-Society Consensus II type C (n = 18; 24.7%) or type D (n = 44; 60.3%), with mean lesion and occluded lengths of 24.8 ± 11.4 and 17.8 ± 11.2 cm, respectively. No procedure-related adverse events occurred, except one distal embolization. The ABI significantly increased after intervention from 0.52 ± 0.12 to 0.80 ± 0.15 (P < 0.0001), with marked improvement in clinical symptoms (Rutherford stage: 2.7 ± 1.0 to 1.1 ± 1.2, P < 0.0001). The mean follow-up period was 31.2 ± 18.0 months, and the primary patency rates at 12 and 24 months were 75.3% and 60.6%, respectively. The independent predictive factors of failed patency were baseline hemoglobin A1c (P = 0.031, hazard radio [HR] 1.51 per 1%), occluded length ≥15 cm (P = 0.036, HR 2.90), and severe dissection (P = 0.033, HR 2.85). Vessel calcification and diameter did not affect primary patency. CONCLUSIONS: CBA is a feasible option for endovascular treatment of FP-CTOs. Diabetic status, occlusion length, and severe dissection after CBA are independent negative predictors of long-term patency.

TodoFirGene: Developing Transcriptome Resources for Genetic Analysis of Abies sachalinensis.

Todo-matsu (Abies sachalinensis) is one of the most important forestry species in Hokkaido, Japan and is distributed from near sea level to the alpine zone. Due to its wide spatial distribution, the species adapts to its environment, displaying phenotypes of ecological relevance. In order to identify candidate genes under natural selection, we collected the transcriptome from the female and male flower, leaf and inner bark. De novo assembly with 34.7 Gb of sequencing reads produced 158,542 transcripts from 69,618 loci, whose estimated coverage reached 95.6% of conserved eukaryotic genes. Homology searches against publicly available databases identified 134,190 (84.6%) transcripts with at least one hit. In total, 28,944 simple sequence repeats (SSRs) and 80,758 single nucleotide variants (SNVs) were detected from 23,570 (14.9%) and 25,366 (16.0%) transcripts, which were valuable for use in genetic analysis of the species. All the annotations were included in a relational database, TodoFirGene, which provides an interface for various queries and homology search, and can be accessed at http://plantomics.mind.meiji.ac.jp/todomatsu/. This database hosts not only the A. sachalinensis transcriptome but also links to the proteomes of 13 other species, allowing a comparative genomic study of plant species.

Comparative transcriptome analysis reveals distinct ethylene-independent regulation of ripening in response to low temperature in kiwifruit.

BACKGROUND: Kiwifruit are classified as climacteric since exogenous ethylene (or its analogue propylene) induces rapid ripening accompanied by ethylene production under positive feedback regulation. However, most of the ripening-associated changes (Phase 1 ripening) in kiwifruit during storage and on-vine occur largely in the absence of any detectable ethylene. This ripening behavior is often attributed to basal levels of system I ethylene, although it is suggested to be modulated by low temperature. RESULTS: To elucidate the mechanisms regulating Phase 1 ripening in kiwifruit, a comparative transcriptome analysis using fruit continuously exposed to propylene (at 20 °C), and during storage at 5 °C and 20 °C was conducted. Propylene exposure induced kiwifruit softening, reduction of titratable acidity (TA), increase in soluble solids content (SSC) and ethylene production within 5 days. During storage, softening and reduction of TA occurred faster in fruit at 5 °C compared to 20 °C although no endogenous ethylene production was detected. Transcriptome analysis revealed 3761 ripening-related differentially expressed genes (DEGs), of which 2742 were up-regulated by propylene while 1058 were up-regulated by low temperature. Propylene exclusively up-regulated 2112 DEGs including those associated with ethylene biosynthesis and ripening such as AcACS1, AcACO2, AcPL1, AcXET1, Acß-GAL, AcAAT, AcERF6 and AcNAC7. Similarly, low temperature exclusively up-regulated 467 DEGS including AcACO3, AcPL2, AcPMEi, AcADH, Acß-AMY2, AcGA2ox2, AcNAC5 and AcbZIP2 among others. A considerable number of DEGs such as AcPG, AcEXP1, AcXET2, Acß-AMY1, AcGA2ox1, AcNAC6, AcMADS1 and AcbZIP1 were up-regulated by either propylene or low temperature. Frequent 1-MCP treatments failed to inhibit the accelerated ripening and up-regulation of associated DEGs by low temperature indicating that the changes were independent of ethylene. On-vine kiwifruit ripening proceeded in the absence of any detectable endogenous ethylene production, and coincided with increased expression of low temperature-responsive DEGs as well as the decrease in environmental temperature. CONCLUSIONS: These results indicate that kiwifruit possess both ethylene-dependent and low temperature-modulated ripening mechanisms that are distinct and independent of each other. The current work provides a foundation for elaborating the control of these two ripening mechanisms in kiwifruit.

Advances in Studies of P-Glycoprotein and Its Expression Regulators.

This review deals with recent advances in studies on P-glycoprotein (P-gp) and its expression regulators, focusing especially on our own research. Firstly, we describe findings demonstrating that the distribution of P-gp along the small intestine is heterogeneous, which explains why orally administered P-gp substrate drugs often show bimodal changes of plasma concentration. Secondly, we discuss the post-translational regulation of P-gp localization and function by the scaffold proteins ezrin, radixin and moesin (ERM proteins), together with recent reports indicating that tissue-specific differences in regulation by ERM proteins in normal tissues might be retained in corresponding cancerous tissues. Thirdly, we review evidence that P-gp activity is enhanced in the process of epithelial-to-mesenchymal transition (EMT), which is associated with cancer progression, without any increase in expression of P-gp mRNA. Finally, we describe two examples in which P-gp critically influences the brain distribution of drugs, i.e., oseltamivir, where low levels of P-gp associated with early development allow oseltamivir to enter the brain, potentially resulting in neuropsychiatric side effects in children, and cilnidipine, where impairment of P-gp function in ischemia allows cilnidipine to enter the ischemic brain, where it exerts a neuroprotective action.