Genetic diversity of submergence stress response in cytoplasms of the Triticum-Aegilops complex.

Genetic diversity in cytoplasmic and nuclear genomes and their interaction affecting adaptive traits is an attractive research subject in plants. We addressed submergence stress response of wheat that has become increasingly important but remained largely uninvestigated. Our primary aim was to disclose cytoplasmic diversity using nucleus-cytoplasm (NC) hybrids possessing a series of heterologous cytoplasms in a common nuclear background. Effects of submergence on seedling emergence and growth from imbibed seeds were studied and compared with euplasmic lines. Marked phenotypic variabilities were observed among both lines, demonstrating divergent cytoplasmic and nuclear effects on submergence response. NC hybrids with cytoplasm of Aegilops mutica showed a less inhibition, indicative of their positive contribution to submergence tolerance, whereas cytoplasms of Aegilops umbellulata and related species caused a greater inhibition. Superoxide dismutase (SOD) activity showed a marked increase accompanied by retardation of seedling growth in a susceptible NC hybrid. The observation suggested that the elevated SOD activity was resulted from a high level of reactive oxygen species accumulated and remained in susceptible seedlings. Taken together, our results point to the usefulness of NC hybrids in further studies needed to clarify molecular mechanisms underlying the nucleus-cytoplasm interaction regulating submergence stress response in wheat.

Mapping QTLs for cold tolerance at germination and the early seedling stage in rice (Oryza sativa L.).

Cold tolerance is an important breeding target in rice production. We studied quantitative trait loci (QTLs) controlling cold tolerance at germination (CTG) and early seedling (CTS) stages, using recombinant inbred lines derived from a japonica × indica cross. CTG was evaluated based on the percentage rate of germination at 15 °C for 12 days after pre-incubation of imbibed seeds at 20 °C for 2 days. For CTS, seven-day-old seedlings grown at 25 °C were subjected to two consecutive periods of three-day cold stress at 4 °C with an intervening eight-day recovery at 25 °C. CTS evaluation was according to an arbitrary five-point rating system at the fifth day of recovery after each stress period. No correlations were found between CTG and CTS, while a weak correlation was detected between CTS after the first and second stress. By the composite interval mapping, five QTLs for CTG explaining 5.7%-9.3% of the total phenotypic variance (PVE) and nine for CTS with PVE of 5.8%-35.6% were detected. Only one of these QTLs was common, indicating growth-stage specificity of cold tolerance. Four of the five QTLs after the second cold stress were different from the ones after the first cold stress. Analysis of variance test showed significant interactions between alleles at the QTL sites and the two stress conditions with respect to the mean CTS scores. A possible involvement of cold acclimation and usefulness of japonica germplasms in breeding for cold tolerance in indica rice was discussed.

Preparation, crystallization and preliminary X-ray diffraction studies of the glycosylated form of human interleukin-23.

Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine composed of p19 and p40 subunits. IL-23 plays crucial roles in the activation, proliferation and survival of IL-17-producing helper T cells which induce various autoimmune diseases. Human p19 and p40 subunits were cloned and coexpressed in N-acetylglucosaminyltransferase I-negative 293S cells, which produce high-mannose-type glycosylated proteins in order to diminish the heterogeneity of modified N-linked glycans. The glycosylated human IL-23 was purified and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were then collected to 2.6 Å resolution. The crystal belonged to space group P6(1) or P6(5), with unit-cell parameters a = b = 108.94, c = 83.79 Å, γ = 120°. Assuming that the crystal contains one molecule per asymmetric unit, the calculated Matthews coefficient was 2.69 Å(3) Da(-1), with a solvent content of 54.2%. The structure was determined by the molecular-replacement method, with an initial R factor of 52.6%. After subsequent rigid-body and positional refinement, the R(work) and R(free) values decreased to 31.4% and 38.7%, respectively.

Profiling of mitochondrial transcriptome in germinating wheat embryos and seedlings subjected to cold, salinity and osmotic stresses.

Mitochondrial functions are potential targets of abiotic stresses that are major environmental factors limiting plant development and productivity. To evaluate mitochondrial responses to abiotic stresses we studied mitochondrial transcriptome profiles at the early stages of wheat development after imbibition under normal and induced stress conditions. Three stresses given were low temperature (4 degrees C), high salinity (0.2 M NaCl) and high osmotic potential (0.3 M mannitol). All these stresses greatly reduced growth but dramatically increased respiration both via the cytochrome and alternative pathways. Macroarray analysis of the mitochondrial transcriptome revealed that most of the changes in transcript levels were stress specific but groups of genes responded commonly to different stresses. Under 3-days continuous stresses, 13 genes showed low temperature specific responses with either up- or down-regulation, while 14 and 23 genes showed responses specific to high salinity and high osmotic potential, respectively. On the other hand, 13 genes showed common responses, among which cob and ccmFn increased their transcript levels while transcripts of the other genes including nad6, atp4 and atp9 decreased. The differential profiles of mitochondrial transcriptome revealed by the macroarray analysis were verified by the quantitative reverse transcriptase PCR analysis. Taken together, three among five nuclear-encoded mitochondria-targeted genes included in the array showed decreases under the stresses, while MnSOD and AOX increased their transcript amounts. Our results indicated the existence of common and different regulatory mechanisms that can sense different abiotic stresses and modulate both nuclear and mitochondrial gene expression in germinating wheat embryos and seedlings.

[Multiple sleep latency test (MSLT) and maintenance of wakefulness test (MWT)].

To define and evaluate sleepiness thoroughly has not yet completely established because of its multidimensional aspects of character. Multiple sleep latency test (MSLT) and maintenance of wakefulness test (MWT) have been the important tools to assess sleepiness in the research and clinical settings, although they have several limitations and pitfalls. The coverage by the National Health Insurance of MSLT, since April 1, 2008, in Japan, so far not MWT, would extend the diagnosis and care of sleep disorders concerning excessive daytime sleepiness including narcolepsy and idiopathic hypersomnia. The relevant and appropriate use of these modalities should be always considered in line with clinical history, medical information and judgment.

Genotyping of the Q locus in wheat by a simple PCR-RFLP method.

The Q locus located on the long arm of chromosome 5A is a key factor in evolution and widespread cultivation of domesticated wheat. The Q locus pleiotropically affects many agronomically important traits including threshability, glume shape and tenacity, rachis fragility and others. Genotyping of the Q locus based on the complex traits is ambiguous due to their multi-genetic control through interactions with the Q locus. To determine the Q locus genotype of wheat accessions possessing A genome, we developed a method based on polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. The Q and q alleles were clearly distinguished by PCR-RFLP analysis at six conserved single nucleotide polymorphisms in common wheat and wild and cultivated einkorn, emmer and timopheevi wheat. The Q locus genotype of Triticum sinskajae, which is one of the einkorn wheat species and exhibits free-threshing trait, was determined to be qq as expected. This simple PCR-RLFP-based genotyping method should serve as a useful tool in studying the origin of Q and thus wheat evolution after domestication and the following widespread cultivation.

Expression profiles of respiratory components associated with mitochondrial biogenesis during germination and seedling growth under normal and restricted conditions in wheat.

Germination of plant embryo is a dynamic phase-changing process that is driven by a rapid increase in mitochondrial respiration. We studied the development of respiratory electron transport pathways and the profiles of their transcript and protein components during this critical period using wheat embryos. Oxygen consumption through both the cytochrome and alternative pathways increased rapidly upon imbibition. Quantitative RT-PCR analysis using specific primers and western blot analysis using specific antibodies suggested that this respiratory burst was supported both by the stored mRNA and protein components and ones synthesized de novo at least in the cytochrome pathway. Dry embryos also contained transcript and protein of alternative oxidase (AOX), but their levels remained constant during the studied period. By contrast, the alternative pathway capacity showed a marked increase when the cytochrome pathway was inhibited by antimycin A and this increase was associated with increased levels of AOX transcript and protein. Our results suggest that mitochondrial biogenesis is accompanied by sequential and differential gene expression and protein accumulation, and that AOX allows the complex I to continue to conserve energy thus to support embryo germination and initial seedling growth in wheat when the cytochrome pathway is restricted.

Increased freezing tolerance in an ABA-hypersensitive mutant of common wheat.

To study role of abscisic acid (ABA) in cold acclimation and cold/freezing tolerance in wheat, we analyzed an ABA-hypersensitive mutant of Triticum aestivum, named ABA27. ABA-hypersensitivity in ABA27 was confirmed by bioassays involving germination and seedling growth and expression analysis of ABA-responsive genes in comparison with the parental cultivar 'Chihoku-komugi' (Chihoku). ABA27 showed significantly increased freezing tolerance in seedlings without cold acclimation. ABA-treated seedlings of ABA27 accumulated more transcripts of ABA-responsive genes Cor/Lea and their putative transcription factor (TF) genes than Chihoku under both normal and low-temperature (LT) conditions. Non-ABA-regulated Cor/Lea transcripts showed higher accumulation in ABA27 also under normal temperature. These results suggest that the elevated ABA sensitivity in ABA27 contributes to the improved freezing tolerance through increased expression of the ABA-regulated LT signal pathway. Based on these and previous results obtained in an ABA-less-sensitive mutant, it is suggested that both positive and negative regulation of ABA response is involved in the basic mechanism of freezing tolerance in wheat.

Sequence diversity and copy number variation of Mutator-like transposases in wheat

Partial transposase-coding sequences of Mutator-like elements (MULEs) were isolated from a wild einkorn wheat, Triticum urartu, by degenerate PCR. The isolated sequences were classified into a MuDR or Class I clade and divided into two distinct subclasses (subclass I and subclass II). The average pair-wise identity between members of both subclasses was 58.8 percent at the nucleotide sequence level. Sequence diversity of subclass I was larger than that of subclass II. DNA gel blot analysis showed that subclass I was present as low copy number elements in the genomes of all Triticum and Aegilops accessions surveyed, while subclass II was present as high copy number elements. These two subclasses seemed uncapable of recognizing each other for transposition. The number of copies of subclass II elements was much higher in Aegilops with the S, Sl and D genomes and polyploid Triticum species than in diploid Triticum with the A genome, indicating that active transposition occurred in S, Sl and D genomes before polyploidization. DNA gel blot analysis of six species selected from three subfamilies of Poaceae demonstrated that only the tribe Triticeae possessed both subclasses. These results suggest that the differentiation of these two subclasses occurred before or immediately after the establishment of the tribe Triticeae.

Mitochondrial biogenesis as revealed by mitochondrial transcript profiles during germination and early seedling growth in wheat.

Germination of imbibed embryos is the initial stage of plant development that is accompanied by the burst of mitochondrial respiration. To understand the process of mitochondrial biogenesis during this critical stage in wheat development, we monitored changes in mitochondrial transcript profiles during the first 3 days by adopting a newly devised macroarray system. The whole experiment was conducted in the dark to avoid influences of photosynthesis. Dry quiescent embryos started respiration rapidly after imbibition and the rate of oxygen uptake increased to peak at the first day followed by a continuous decrease until the third day under this condition. Both the cytochrome and alternative electron transport pathways appeared to contribute to this initial burst. Shoot and root growth was also remarkable during this period. Mitochondrial transcriptome was studied by macroarray analysis using 28 mitochondrial protein-coding genes, 4 nuclear encoded mitochondria-targeted genes and 2 nuclear genes as control. All transcripts were present in dry embryos at different initial levels, and a large variability was observed in their abundance among individual genes throughout the tested period. Gene expression was categorized into four clusters according to the profiles of individual transcript accumulation. A majority of the genes encoding subunits of the respiratory complexes belonged to two major clusters, the time course of transcript accumulation of one cluster agreeing with that of respiratory development and the other remaining at high constant levels. The macroarray system devised in this study should be useful in monitoring mitochondrial biogenesis under various growth conditions and at different developmental stages in cereals.

Overexpression of wheat alternative oxidase gene Waox1a alters respiration capacity and response to reactive oxygen species under low temperature in transgenic Arabidopsis.

Under low temperature conditions, the cytochrome pathway of respiration is repressed and reactive oxygen species (ROS) are produced in plants. Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for the cyanide-insensitive and salicylhydroxamic acid-sensitive respiration. To study functions of wheat AOX genes under low temperature, we produced transgenic Arabidopsis by introducing Waox1a expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis thaliana. The enhancement of endogenous AOX1a expression via low temperature stress was delayed in the transgenic Arabidopsis. Recovery of the total respiration activity under low temperature occurred more rapidly in the transgenic plants than in the wild-type plants due to a constitutively increased alternative pathway capacity. Levels of ROS decreased in the transgenic plants under low temperature stress. These results support the hypothesis that AOX alleviates oxidative stress when the cytochrome pathway of respiration is inhibited under abiotic stress conditions.

Ramsay-Hunt syndrome with vesicular stomatitis in a 4-year-old infant.

Ramsay-Hunt syndrome (RHS) usually affects adults, but rare cases of preschool children with RHS have been reported. We report a case of RHS in a healthy 4-year-old girl. At the age of 4 years and 5 months, she complained of pain in her mouth and herpes zoster vesicles were noted on the left soft palate and tongue without left pinna, and complete left facial paralysis subsequently developed. She was treated with acyclovir and steroids. Six months later, her facial paralysis had almost fully resolved.

Differential regulation of transcript accumulation and alternative splicing of a DREB2 homolog under abiotic stress conditions in common wheat.

A number of cold responsive (Cor)/late embryogenesis abundant (Lea) genes are induced by both low temperature (LT) and dehydration. To understand the molecular basis of cold acclimation and its relationship with drought stress response in wheat seedlings, we isolated a DREB2 homolog Wdreb2, which is the candidate gene for a transcription factor of the Cor/Lea genes. The Wdreb2 expression was activated by cold, drought, salt and exogenous ABA treatment. Detailed expression studies of Wdreb2 indicated the involvement of two distinct pathways of its activation, a drought and salt stress-responsive pathway and a cold-responsive pathway. The transient expression analysis showed that the Wrab19 expression was directly activated by the WDREB2 transcription factor in wheat cells. Three transcript forms of Wdreb2 (Wdreb2alpha, Wdreb2beta and Wdreb2gamma) were produced through alternative splicing. Under drought and salt stress conditions, the amount of the Wdreb2beta form remained fairly constant during 24-hour treatment, while those of the Wdreb2alpha and Wdreb2gamma forms showed transient increases. On the other hand, the LT treatment resulted in increased transcript levels of all three forms of Wdreb2. Thus, under the LT and drought/salt stress conditions the amount of the WDREB2 transcription factors in wheat is differentially controlled by the level of transcription and alternative splicing.

Improvement of freezing tolerance in tobacco plants expressing a cold-responsive and chloroplast-targeting protein WCOR15 of wheat.

Cold acclimation, an adaptive process for developing freezing tolerance in over-wintering plants, is associated with increased expression levels of a series of cold-responsive (Cor)/late embryogenesis abundant (Lea) genes. To investigate the function of Wcor15, a member of the wheat Cor/Lea gene family, for improvement of freezing tolerance, two types of transgenic tobacco lines expressing Wcor15-containing chimeric genes were produced and characterized. Immunoblot and gene expression analyses of a transgenic tobacco line expressing the Wcor15-GFP fusion gene under control of the CaMV35S promoter showed transport and abundant accumulation of the WCOR15 protein in the stromal compartment of the chloroplasts. The 5' upstream region of Wcor15 induced expression of the GFP reporter gene under low-temperature conditions in the transgenic tobacco. Both transgenic lines expressing the Wcor15-GFP fusion gene showed a similar and significantly improved level of freezing tolerance compared with the wild-type tobacco plants. Our results demonstrate that the induced expression of the wheat Wcor15 gene positively contributes to the development of freezing tolerance in the heterologous tobacco plants.

Novel cytoprotective mechanism of anti-parkinsonian drug deprenyl: PI3K and Nrf2-derived induction of antioxidative proteins.

Neuroprotection has received considerable attention as a strategy for the treatment of Parkinson's disease (PD). Deprenyl (Selegiline) is a promising candidate for neuroprotection; however, its cytoprotective mechanism has not been fully clarified. Here, we report a novel cytoprotective mechanism of deprenyl involving PI3K and Nrf2-mediated induction of oxidative stress-related proteins. Deprenyl increased the expression of HO-1, PrxI, TrxI, TrxRxI, gammaGCS, and p62/A170 in SH-SY5Y cells. Deprenyl also induced the nuclear accumulation of Nrf2 and increased the binding activity of Nrf2 to the enhancer region of human genomic HO-1. The Nrf2-mediated induction of antioxidative molecules was controlled by PI3K. Indeed, furthermore, neurotrophin receptor TrkB was identified as an upstream signal for PI3K-Nrf2 activation by deprenyl. These results suggest that the cytoprotective effect of deprenyl is, in part, dependent on Nrf2-mediated induction of antioxidative proteins, suggesting that activation of the PI3K-Nrf2 system may be a useful therapeutic strategy for PD.

Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome.

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon-intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Differential and coordinated expression of Cbf and Cor/Lea genes during long-term cold acclimation in two wheat cultivars showing distinct levels of freezing tolerance.

The cold acclimation process in plants is primarily regulated through the signal transduction pathways that lead to the induction and enhancement of expression of different sets of Cor/Lea genes. Winter wheat 'Mironovskaya 808' (M808) exhibited a much higher level of freezing tolerance than spring wheat 'Chinese Spring' (CS), and the difference became clearer after the long-term cold acclimation. To understand the molecular basis of this cultivar difference, we isolated two CBF/DREB1 homologs, Wcbf2, which are the candidate gene for a transcription factor of the Cor/Lea genes. Expression of the Wcbf2 gene was induced rapidly by low temperature (LT) and drought but not by abscisic acid (ABA). The gene expression was temporal and at least twice up-regulated by LT. The first up-regulation occurred within 1-4 h, which might correspond to the rapid response to LT, while the second up-regulation occurred during 2-3 weeks of cold acclimation. After the second up-regulation, the amount of Wcbf2 transcript greatly decreased in CS, while it increased again in M808 after 4 weeks until 9 weeks (end of the test period). The maintenance of this high level of the Wcbf2 transcript might represent the long-term effect of cold acclimation. The activation of Cor/Lea genes followed the accumulation of Wcbf2 transcript suggested direct involvement of the Wcbf2 gene in the induction and enhancement of the Cor/Lea gene expression. The cultivar difference in freezing tolerance developed during different stages of cold acclimation can be at least partly explained by the differential and coordinated regulation of the predicted Cor/Lea gene signal transduction pathway that is mediated by the CBF/DREB1 transcription factors in common wheat.

Intragenic diversity and functional conservation of the three homoeologous loci of the KN1-type homeobox gene Wknox1 in common wheat.

Common wheat represents a typical allohexaploid species and provides a good experimental system for studying genomic alterations associated with allopolyploidization. We studied three homoeologous loci of wheat Wknox1 gene, which is highly homologous to the knotted1 (kn1)-like homeobox (KNOX) genes functioning at shoot apical meristems (SAM). Wknox1 transcripts were detected in SAM, and its overexpression caused abnormal leaf morphology with occasional ectopic leaves in transgenic tobacco plants. A comparative study of the three Wknox1 genomic sequences revealed accumulation of a large number of mutations including insertions and deletions, particularly in the fourth intron and the 5'-upstream region. Some structural mutations including MITE-insertions were allocated in the evolutionary lineage of the wheat genome, suggesting that they occurred at all stages of wheat evolution. A mutation rate was the highest in the Wknox1b locus, which is consistent with the known highest degree of differentiation in the B genome. Despite the structural differentiation, all three Wknox1 homoeologs showed an identical expression pattern in wheat and their promoter regions induced the conserved expression pattern in transgenic tobacco plants. A potential of the intragenic diversity in homoeologs of essential genes as a tool for studying the genome evolution associated with allopolyploidization was discussed.