High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

<p>Abstract</p> <p>Background</p> <p>During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective.</p> <p>Results</p> <p>In this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the <it>p53 </it>gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in <it>p53</it>, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the <it>atr </it>and <it>atm </it>genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature.</p> <p>Conclusions</p> <p>These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.</p

Widely Targeted Metabolomics Based on Large-Scale MS/MS Data for Elucidating Metabolite Accumulation Patterns in Plants

Metabolomics is an ‘omics’ approach that aims to analyze all metabolites in a biological sample comprehensively. The detailed metabolite profiling of thousands of plant samples has great potential for directly elucidating plant metabolic processes. However, both a comprehensive analysis and a high throughput are difficult to achieve at the same time due to the wide diversity of metabolites in plants. Here, we have established a novel and practical metabolomics methodology for quantifying hundreds of targeted metabolites in a high-throughput manner. Multiple reaction monitoring (MRM) using tandem quadrupole mass spectrometry (TQMS), which monitors both the specific precursor ions and product ions of each metabolite, is a standard technique in targeted metabolomics, as it enables high sensitivity, reproducibility and a broad dynamic range. In this study, we optimized the MRM conditions for specific compounds by performing automated flow injection analyses with TQMS. Based on a total of 61,920 spectra for 860 authentic compounds, the MRM conditions of 497 compounds were successfully optimized. These were applied to high-throughput automated analysis of biological samples using TQMS coupled with ultra performance liquid chromatography (UPLC). By this analysis, approximately 100 metabolites were quantified in each of 14 plant accessions from Brassicaceae, Gramineae and Fabaceae. A hierarchical cluster analysis based on the metabolite accumulation patterns clearly showed differences among the plant families, and family-specific metabolites could be predicted using a batch-learning self-organizing map analysis. Thus, the automated widely targeted metabolomics approach established here should pave the way for large-scale metabolite profiling and comparative metabolomics

Activation of Gibberellin Biosynthesis and Response Pathways by Low Temperature during Imbibition of Arabidopsis thaliana Seeds

Exposure of imbibed seeds to low temperature (typically 4°C) is widely used to break seed dormancy and to improve the frequency of germination. However, the mechanism by which temperature accelerates germination is largely unknown. Using DNA microarray and gas chromatography–mass spectrometry analyses, we found that a subset of gibberellin (GA) biosynthesis genes were upregulated in response to low temperature, resulting in an increase in the level of bioactive GAs and transcript abundance of GA-inducible genes in imbibed Arabidopsis thaliana seeds. Using a loss-of-function mutant, the cold-inducible GA biosynthesis gene, AtGA3ox1, was shown to play an essential role in mediating the effect of low temperature. Besides temperature, AtGA3ox1 also is positively regulated by active phytochrome and negatively regulated by GA activity. We show that both red light and GA deficiency act in addition to low temperature to elevate the level of AtGA3ox1 transcript, indicating that multiple signals are integrated by the AtGA3ox1 gene to control seed germination. When induced by low temperature, AtGA3ox1 mRNA was detectable by in situ RNA hybridization in an additional set of cell types relative to that in red light–induced seeds. Our results illustrate that the GA biosynthesis and response pathways are activated during seed imbibition at low temperature and suggest that the cellular distribution of bioactive GAs may be altered under different light and temperature conditions

Gibberellin Biosynthesis and Response during Arabidopsis Seed Germination

The hormone-mediated control of plant growth and development involves both synthesis and response. Previous studies have shown that gibberellin (GA) plays an essential role in Arabidopsis seed germination. To learn how GA stimulates seed germination, we performed comprehensive analyses of GA biosynthesis and response using gas chromatography–mass spectrometry and oligonucleotide-based DNA microarray analysis. In addition, spatial correlations between GA biosynthesis and response were assessed by in situ hybridization. We identified a number of transcripts, the abundance of which is modulated upon exposure to exogenous GA. A subset of these GA-regulated genes was expressed in accordance with an increase in endogenous active GA levels, which occurs just before radicle emergence. The GA-responsive genes identified include those responsible for synthesis, transport, and signaling of other hormones, suggesting the presence of uncharacterized crosstalk between GA and other hormones. In situ hybridization analysis demonstrated that the expression of GA-responsive genes is not restricted to the predicted site of GA biosynthesis, suggesting that GA itself, or GA signals, is transmitted across different cell types during Arabidopsis seed germination

CYP707A1 and CYP707A2, Which Encode Abscisic Acid 8′-Hydroxylases, Are Indispensable for Proper Control of Seed Dormancy and Germination in Arabidopsis

Endogenous abscisic acid (ABA) levels are regulated by both biosynthesis and catabolism of the hormone. ABA 8′-hydroxylase is considered to be the key catabolic enzyme in many physiological processes. We have previously identified that four members of the Arabidopsis (Arabidopsis thaliana) CYP707A gene family (CYP707A1 to CYP707A4) encode ABA 8′-hydroxylases, and that the cyp707a2 mutants showed an increase in ABA levels in dry and imbibed seeds. In this study, we showed that the cyp707a1 mutant accumulated ABA to higher levels in dry seeds than the cyp707a2 mutant. Expression analysis showed that the CYP707A1 was expressed predominantly during mid-maturation and was down-regulated during late-maturation. Concomitantly, the CYP707A2 transcript levels increased from late-maturation to mature dry seed. Phenotypic analysis of single and double cyp707a mutants indicates that the CYP707A1 is important for reducing ABA levels during mid-maturation. On the other hand, CYP707A2 is responsible for the regulation of ABA levels from late-maturation to germination. Moreover, CYP707A1 and CYP707A3 were also shown to be involved in postgermination growth. Spatial expression analysis suggests that CYP707A1 was expressed predominantly in embryo during mid-maturation, whereas CYP707A2 expression was detected in both embryo and endosperm from late-maturation to germination. Our results demonstrate that each CYP707A gene plays a distinct role during seed development and postgermination growth

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana

10.1073/pnas.2017890118Proceedings of the National Academy of Sciences of the United States of America11822e201789011

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by -40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms