[Show abstract][Hide abstract] ABSTRACT: The American alligator (Alligator mississippiensis) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD.
Our characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B, and transcription factor C/EBPA. Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX. Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9, and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted.
Gonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.
[Show abstract][Hide abstract] ABSTRACT: In land plants, there are two types of male gametes: one is a non-motile sperm cell which is delivered to the egg cell by
a pollen tube, the other is a motile sperm cell with flagella. The molecular mechanism underlying the sexual reproduction
with the egg and pollen-delivered sperm cell is well understood from studies using model plants such as Arabidopsis and rice. On the other hand, the sexual reproduction with motile sperm has remained poorly characterized, due to the lack
of suitable models. Marchantia polymorpha L. is a model basal land plant with sexual reproduction involving an egg cell and bi-flagellated motile sperm. To understand
the differentiation process of plant motile sperm, we analyzed the gene expression profile of developing antheridia of M. polymorpha. We performed RNA-sequencing experiments and compared transcript profiles of the male sexual organ (antheridiophore and antheridium
contained therein), female sexual organ (archegoniophore), and a vegetative organ (thallus). Transcriptome analysis showed
that the antheridium expresses nearly half of the protein-coding genes predicted in the genome, but it also has unique features.
The antheridium transcriptome shares some common features with male gamete transcriptomes of angiosperms and animals, and
homologs of genes involved in male gamete formation and function in angiosperms and animals were identified. In addition,
we showed that some of them had distinct expression patterns in the spermatogenous tissue of developing antheridia. This study
provides a transcriptional framework on which to study the molecular mechanism of plant motile sperm development in M. polymorpha as a model.
No preview · Article · Feb 2016 · Plant and Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: Magnesium (Mg) is an essential macronutrient, functioning as both a cofactor of many enzymes and as a component of Chl. Mg
is abundant in plants; however, further investigation of the Mg transporters involved in Mg uptake and distribution is needed.
Here, we isolated an Arabidopsis thaliana mutant sensitive to high calcium (Ca) conditions without Mg supplementation. The causal gene of the mutant encodes MRS2-4,
an Mg transporter. MRS2-4 single mutants exhibited growth defects under low Mg conditions, whereas an MRS2-4 and MRS2-7 double mutant exhibited growth defects even under normal Mg concentrations. Under normal Mg conditions, the Mg concentration
of the MRS2-4 mutant was lower than that of the wild type. The transcriptome profiles of mrs2-4-1 mutants under normal conditions were similar to those of wild-type plants grown under low Mg conditions. In addition, both
mrs2-4 and mrs2-7 mutants were sensitive to high levels of Mg. These results indicate that both MRS2-4 and MRS2-7 are essential for Mg homeostasis,
even under normal and high Mg conditions. MRS2-4–green fluorescent protein (GFP) was mainly detected in the endoplasmic reticulum.
These results indicate that these two MRS2 transporter genes are essential for the ability to adapt to a wide range of environmental
No preview · Article · Jan 2016 · Plant and Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: The cladoceran crustacean Daphnia pulex produces female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable external stimuli, it produces male offspring (environmental sex determination: ESD). We recently established an innovative system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be controlled simply by changes in the photoperiod: the long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we demonstrated that de novo methyl farnesoate (MF) synthesis is necessary for male offspring production. These results indicate the key role of innate MF signaling as a conductor between external environmental stimuli and the endogenous male developmental pathway. Despite these findings, the molecular mechanisms underlying up- and downstream signaling of MF have not yet been well elucidated in D. pulex.
To elucidate up- and downstream events of MF signaling during sex determination processes, we compared the transcriptomes of daphnids reared under the long-day (female) condition with short-day (male) and MF-treated (male) conditions. We found that genes involved in ionotropic glutamate receptors, known to mediate the vast majority of excitatory neurotransmitting processes in various organisms, were significantly activated in daphnids by the short-day condition but not by MF treatment. Administration of specific agonists and antagonists, especially for the N-methyl-D-aspartic acid (NMDA) receptor, strongly increased or decreased, respectively, the proportion of male-producing mothers. Moreover, we also identified genes responsible for male production (e.g., protein kinase C pathway-related genes). Such genes were generally shared between the short-day reared and MF-treated daphnids.
We identified several candidate genes regulating ESD which strongly suggests that these genes may be essential factors for male offspring production as an upstream regulator of MF signaling in D. pulex. This study provides new insight into the fundamental mechanisms underlying how living organisms alter their phenotypes in response to various external environments.
[Show abstract][Hide abstract] ABSTRACT: Calcium (Ca) deficiency symptoms in plants often occur in agriculture; however, little is known about the mechanisms for adaptation to low-Ca conditions. To understand the mechanisms, we screened for Arabidopsis thaliana (L.) Heynh mutants sensitive to low Ca. Here, we describe one of the mutants, lcs1-1, isolated from the screen. The relative shoot growth of the mutant was reduced under the low-Ca conditions compared with the wild-type plants. Genetic mapping and genome resequencing revealed that lcs1-1 has one nonsynonymous mutation in the region of the chromosome responsible for the phenotype. The mutation is in Pleiotropic Regulatory Locus 1 (PRL1). An allelism test between lcs1-1 and a T-DNA inserted allele of prl1 demonstrates that the causal gene of lcs1-1 is PRL1. It has been reported that PRL1 is involved in sugar metabolism; however, the involvement of PRL1 in low-Ca tolerance has not been reported. Our results suggest a new insight connecting sugar metabolism with a mechanism for low-Ca tolerance in plants.
No preview · Article · Sep 2015 · Soil Science and Plant Nutrition
[Show abstract][Hide abstract] ABSTRACT: Human papillomavirus type 16 (HPV16) is a major cause of cervical cancer. We previously demonstrated that C-to-T and G-to-A hypermutations accumulated in the HPV16 genome by APOBEC3 expression in vitro. To investigate in vivo characteristics of hypermutation, differential DNA denaturation-PCR (3D-PCR) was performed using three clinical specimens obtained from HPV16-positive cervical dysplasia, and detected hypermutation from two out of three specimens. One sample accumulating hypermutations in both E2 and the long control region (LCR) was further subjected to Next-Generation Sequencing, revealing that hypermutations spread across the LCR and all early genes. Notably, hypermutation was more frequently observed in the LCR, which contains a viral replication origin and the early promoter. APOBEC3 expressed abundantly in an HPV16-positive cervix, suggesting that single-stranded DNA exposed during viral replication and transcription may be efficient targets for deamination. The results further strengthen a role of APOBEC3 in introducing HPV16 hypermutation in vivo.
[Show abstract][Hide abstract] ABSTRACT: Chemical communication is essential for the coordination of complex organisation in ant societies. Recent comparative genomic approaches have revealed that chemosensory genes are diversified in ant lineages, and suggest that this diversification is crucial for social organisation. However, how such diversified genes shape the peripheral chemosensory systems remains unknown. In this study, we annotated and analysed the gene expression profiles of chemosensory proteins (CSPs), which transport lipophilic compounds toward chemosensory receptors in the carpenter ant, Camponotus japonicus. Transcriptome analysis revealed 12 CSP genes and phylogenetic analysis showed that 3 of these are lineage-specifically expanded in the clade of ants. RNA sequencing and real-time quantitative polymerase chain reaction revealed that, among the ant specific CSP genes, two of them (CjapCSP12 and CjapCSP13) were specifically expressed in the chemosensory organs and differentially expressed amongst ant castes. Furthermore, CjapCSP12 and CjapCSP13 had a ratio of divergence at non-synonymous and synonymous sites (dN/dS) greater than 1, and they were co-expressed with CjapCSP1, which is known to bind cuticular hydrocarbons. Our results suggested that CjapCSP12 and CjapCSP13 were functionally differentiated for ant-specific chemosensory events, and that CjapCSP1, CjapCSP12, and CjapCSP13 work cooperatively in the antennal chemosensilla of worker ants.
Full-text · Article · Aug 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Plant developmental processes are co-ordinated with the status of cell metabolism, not only in mitochondria but also in plastids.
In Arabidopsis thaliana, succinic semialdehyde (SSA), a GABA shunt metabolite, links the specific mitochondrial metabolic pathway to shoot development.
To understand the mechanism of SSA-mediated development, we isolated a succinic semialdehyde dehydrogenase (ssadh) suppressor mutant, affected in its ability to catalyze SSA to succinic acid. We found that pleiotropic developmental phenotypes
of ssadh are suppressed by a mutation in GLUTAMATE-1-SEMIALDEHYDE 2, 1-AMINOMUTASE 2 (GSA2), which encodes a plastidial enzyme converting glutatamate-1-semialdehyde to 5-aminolevulinic acid (5-ALA). In addition,
a mutation in either HEMA1 or GSA1, two other enzymes for 5-ALA synthesis, also suppressed ssadh fully and partially, respectively. Furthermore, exogenous application of 5-ALA and SSA disturbed leaf development. These
results suggest that metabolism in both mitochondria and plastids affect shoot development.
No preview · Article · Jun 2015 · Plant and Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: The low frequency of meiotic recombination in chromosomal regions other than hotspots is a general obstacle to efficient breeding. A number of active genes are present in recombination-repressed centromeric regions in higher eukaryotes, suggesting that suppression of meiotic recombination prevents shuffling of genes within a centromeric region. In this study, by using an inter-subspecific cross of Oryza sativa L., we show that modification of inactive chromatin states by either genetic or chemical inhibition of chromatin modifying proteins induced changes in both the position of meiotic recombination and, unexpectedly, the pattern of segregation distortion of parental alleles. Antisense knockdown of rice homologues of DECREASE IN DNA METHYLATION1, which is required for the maintenance of heterochromatin in Arabidopsis thaliana, induced a recombination hotspot in a centromeric region accompanied by a steep increase in the proportion of heterozygotes. Our results describe a previously undocumented phenomenon in which artificial chromatin modification could be used to change the pattern of segregation distortion in rice and open up novel possibilities for efficient crop breeding.
No preview · Article · Apr 2015 · Molecular Breeding
[Show abstract][Hide abstract] ABSTRACT: Maternal investment for offspring’s growth and survival is widespread among diverse organisms [1, 2 and 3]. Vertical symbiont transmission via maternal passage is also pivotal for offspring’s growth and survival in many organisms [4, 5 and 6]. Hence, it is expected that vertical symbiont transmission may coevolve with various organismal traits concerning maternal investment in offspring. Here we report a novel phenotypic syndrome entailing morphological, histological, behavioral, and ecological specializations for maternal investment and vertical symbiont transmission in stinkbugs of the family Urostylididae [7, 8 and 9]. Adult females develop huge ovaries exaggerated for polysaccharide excretion, possess novel ovipositor-associated organs for vertical transmission of a bacterial symbiont (“Candidatus Tachikawaea gelatinosa”), and lay eggs covered with voluminous symbiont-supplemented jelly. Newborns hatch in midwinter, feed solely on the jelly, acquire the symbiont, and grow during winter. In spring, the insects start feeding on plant sap, wherein the symbiont localizes to a specialized midgut region and supplies essential amino acids deficient in the host’s diet. The reduced symbiont genome and host-symbiont cospeciation indicate their obligate association over evolutionary time. Experimental deprivation of the jelly results in nymphal mortality, whereas restoration of the jelly leads to recovered nymphal growth, confirming that the jelly supports nymphal growth in winter. Chemical analyses demonstrate that the galactan-based jelly contains a sufficient quantity of amino acids to sustain nymphal growth to the third instar. The versatile biological roles of the symbiont-containing egg-covering jelly highlight intricate evolutionary interactions between maternal resource investment and vertical symbiont transmission, which are commonly important for offspring’s growth, survival, and ecological adaptation.
[Show abstract][Hide abstract] ABSTRACT: Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gβ mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.
[Show abstract][Hide abstract] ABSTRACT: The ciliate Paramecium bursaria harbors several hundred cells of the green-alga Chlorella sp. in their cytoplasm. Irrespective of the mutual relation between P. bursaria and the symbiotic algae, both cells retain the ability to grow without the partner. They can easily reestablish endosymbiosis when put in contact with each other. Consequently, P. bursaria is an excellent model for studying cell-cell interaction and the evolution of eukaryotic cells through secondary endosymbiosis between different protists. Despite the importance of this organism, no genomic resources have been identified for P. bursaria to date. This investigation compared gene expressions through RNA-Seq analysis and de novo transcriptome assembly of symbiont-free and symbiont-bearing host cells.
To expedite the process of gene discovery related to the endosymbiosis, we have undertaken Illumina deep sequencing of mRNAs prepared from symbiont-bearing and symbiont-free P. bursaria cells. We assembled the reads de novo to build the transcriptome. Sequencing using Illumina HiSeq2000 platform yielded 232.3 million paired-end sequence reads. Clean reads filtered from the raw reads were assembled into 68,175 contig sequences. Of these, 10,557 representative sequences were retained after removing Chlorella sequences and lowly expressed sequences. Nearly 90% of these transcript sequences were annotated by similarity search against protein databases. We identified differentially expressed genes in the symbiont-bearing P. bursaria cells relative to the symbiont-free cells, including heat shock 70 kDa protein and glutathione S-transferase.
This is the first reported comprehensive sequence resource of Paramecium - Chlorella endosymbiosis. Results provide some keys for the elucidation of secondary endosymbiosis in P. bursaria. We identified P. bursaria genes that are differentially expressed in symbiont-bearing and symbiont-free conditions.
[Show abstract][Hide abstract] ABSTRACT: Sex chromosomes turn over rapidly in some taxonomic groups, where closely related species have different sex chromosomes. Although there are many examples of sex chromosome turnover, we know little about the functional roles of sex chromosome turnover in phenotypic diversification and genomic evolution. The sympatric pair of Japanese threespine stickleback (Gasterosteus aculeatus) provides an excellent system to address these questions: the Japan Sea species has a neo-sex chromosome system resulting from a fusion between an ancestral Y chromosome and an autosome, while the sympatric Pacific Ocean species has a simple XY sex chromosome system. Furthermore, previous quantitative trait locus (QTL) mapping demonstrated that the Japan Sea neo-X chromosome contributes to phenotypic divergence and reproductive isolation between these sympatric species. To investigate the genomic basis for the accumulation of genes important for speciation on the neo-X chromosome, we conducted whole genome sequencing of males and females of both the Japan Sea and the Pacific Ocean species. No substantial degeneration has yet occurred on the neo-Y chromosome, but the nucleotide sequence of the neo-X and the neo-Y has started to diverge, particularly at regions near the fusion. The neo-sex chromosomes also harbor an excess of genes with sex-biased expression. Furthermore, genes on the neo-X chromosome showed higher non-synonymous substitution rates than autosomal genes in the Japan Sea lineage. Genomic regions of higher sequence divergence between species, genes with divergent expression between species, and QTL for inter-species phenotypic differences were found not only at the regions near the fusion site, but also at other regions along the neo-X chromosome. Neo-sex chromosomes can therefore accumulate substitutions causing species differences even in the absence of substantial neo-Y degeneration.
[Show abstract][Hide abstract] ABSTRACT: Background
Although the infection rate of Helicobacter suis is significantly lower than that of Helicobacter pylori, the H. suis infection is associated with a high rate of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. In addition, in vitro cultivation of H. suis remains difficult, and some H. suis-infected patients show negative results on the urea breath test (UBT).Materials and Methods
Female C57BL/6J mice were orally inoculated with mouse gastric mucosal homogenates containing H. suis strains TKY or SNTW101 isolated from a cynomolgus monkey or a patient suffering from nodular gastritis, respectively. The high-purity chromosomal DNA samples of H. suis strains TKY and SNTW101 were prepared from the infected mouse gastric mucosa. The SOLiD sequencing of two H. suis genomes enabled comparative genomics of 20 Helicobacter and 11 Campylobacter strains for the identification of the H. suis-specific nucleotide sequences.ResultsOral inoculation with mouse gastric mucosal homogenates containing H. suis strains TKY and SNTW101 induced gastric MALT lymphoma and the formation of gastric lymphoid follicles, respectively, in C57BL/6J mice. Two conserved nucleotide sequences among six H. suis strains were identified and were used to design diagnostic PCR primers for the detection of H. suis.Conclusions
There was a strong association between the H. suis infection and gastric diseases in the C57BL/6 mouse model. PCR diagnosis using an H. suis-specific primer pair is a valuable method for detecting H. suis in gastric biopsy specimens.
[Show abstract][Hide abstract] ABSTRACT: Endomembrane organization is important for various aspects of cell physiology, including membrane protein trafficking. To explore the molecular mechanisms regulating the trafficking of plasma membrane-localized proteins in plants, we screened for Arabidopsis mutants with defective localization of GFP-NIP5;1. Fluorescence imaging-based screening led to the isolation of a mutant, which accumulated abnormal intracellular aggregates labeled by GFP-NIP5;1. The aggregates appeared in epidermal cells in the root elongation zone and included the trans-Golgi network/early endosomes. Rough mapping and whole-genome sequencing identified the mutant as an allele of UDP-glucose 4-epimerase (uge) 4/root hair defective (rhd) 1/root epidermal bulgar (reb) 1, which was originally defined as a cell wall mutant. The responsible gene encodes UDP-glucose 4-epimerase 4 (UGE4), which functions in the biosynthesis of d-galactose, especially for the synthesis of the cell wall polysaccharide xyloglucan and arabinogalactan proteins (AGPs). The endomembrane aggregates in the mutants were absent in the presence of d-galactose, indicative of a requirement for a d-galactose-containing component in endomembrane organization. Genetic and pharmacological analyses suggested that the aggregates were not caused by the disruption of cell wall polysaccharides or the cytoskeleton. Overall, our results suggest that UGE4 activity in d-galactose synthesis is required for the structure of cell wall polysaccharides and endomembrane organization.
Full-text · Article · Dec 2013 · Plant and Cell Physiology