Metabolic consequences of knocking outUGT85B1, the gene encoding the glucosyltransferase required for synthesis of dhurrin inSorghum bicolor(L. Moench)
Cecilia K. BlomstedtNatalie H. O’DonnellNanna BjarnholtAlan NealeJohn D. HamillBirger Lindberg MøllerRoslyn M. Gleadow
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Many important food crops produce cyanogenic glucosides as natural defense compounds to protect against herbivory or pathogen attack. It has also been suggested that these nitrogen-based secondary metabolites act as storage reserves of nitrogen. In sorghum, three key genes, CYP79A1 , CYP71E1 and UGT85B1 , encode two Cytochrome P450s and a glycosyltransferase, respectively, the enzymes essential for synthesis of the cyanogenic glucoside dhurrin. Here, we report the use of targeted induced local lesions in genomes (TILLING) to identify a line with a mutation resulting in a premature stop codon in the N-terminal region of UGT85B1 . Plants homozygous for this mutation do not produce dhurrin and are designated tcd2 ( totally cyanide deficient 2 ) mutants. They have reduced vigor, being dwarfed, with poor root development and low fertility. Analysis using liquid chromatography–mass spectrometry (LC-MS) shows that tcd2 mutants accumulate numerous dhurrin pathway-derived metabolites, some of which are similar to those observed in transgenic Arabidopsis expressing the CYP79A1 and CYP71E1 genes. Our results demonstrate that UGT85B1 is essential for formation of dhurrin in sorghum with no co-expressed endogenous UDP-glucosyltransferases able to replace it. The tcd2 mutant suffers from self-intoxication because sorghum does not have a feedback mechanism to inhibit the initial steps of dhurrin biosynthesis when the glucosyltransferase activity required to complete the synthesis of dhurrin is lacking. The LC-MS analyses also revealed the presence of metabolites in the tcd2 mutant which have been suggested to be derived from dhurrin via endogenous pathways for nitrogen recovery, thus indicating which enzymes may be involved in such pathways.Keywords:
Glucosyltransferases
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Vitaceae
Abstract Targeting Induced Local Lesions In Genomes (TILLInG) is a reverse genetic strategy, which combines traditional mutagenesis with high-throughput molecular biology protocols. TILLInG was first established in Arabidopsis thaliana when other reverse genetic strategies did not produce acceptable results. TILLInG libraries have been established in several species and more are in progress. Time must be invested in creating a good TILLInG population, such that the mutation frequency is balanced with lethality. In addition, the DNA must be stable. The number of examples of the usefulness of TILLInG libraries is continually growing. While there are some disadvantages, most can be overcome. The flexibility of TILLInG has established it as a useful strategy with great potential to advance agriculture.
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Targeting Induced Local Lesion in Genomes(TILLING) is a reverse genetic strategy that uses traditional chemical mutagenesis methods to create libraries of mutagenized individuals later subjected to high throughput screens for the discovery of an allelic series of induced point mutations in target genes.Usually,TILLING technique consisted of applications of restriction enzyme digestion and Polymerase Chain Reaction(PCR) as well as infrared fluorescence detection of IRD700 nm and IRD 800 nm on acrylamide electrophresis by Licor-segue genetic analyser.Differing from TILLING only for induced mutations by chemical mutagens,Ecotilling stemming from traditional TILLING is generally used for identification of natural mutations and an obvious difference between TILLING and Ecotilling is the mean of establishing DNA pools.With the successful application of TILLING and Ecotilling in model plants such as Arabidopsis thaliana ect.,more and more studies are focusing on the improvement of plants with large genome size.In this review,application of TILLING in Triticeae crops was reviewed and compared with sharply higher percentage of mutation than those of other reported plants.Therefore,TILLING will be promisingly used as a common method to improve characters of Triticeae crops,especially bread wheat(Triticum eastivum L.).
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TILLING (Targeting Induced Local Lesions IN Genomes) and ECOTILLING are methods used in detecting induced or naturally occurring mutations in many species. High-throughput TILLING allows the rapid, easy and cost-effective discovery of induced point mutations in populations of chemically mutagenized individuals. The use of the TILLING technique to survey natural variation in genes is called ECOTILLING. TILLING and ECOTILLING have recently been used for the detection of both induced mutation and natural DNA polymorphism. In this review,we illustrate how TILLING and ECOTILLING methods can be employed for discovering mutations.
Key words: TILLING, ECOTILLING, mutation, natural.
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TILLING(Targeting induced local lesions in genomes) technology is a nove1 reverse genetics strategy that provides a completely solutions for high throughput and low cost discovery of induced point mutations in populations of chemically mutagenized individuals.The TILLING methodology and its technological routine were introduced.Meanwhile,some problems in TILLING were presented.
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This chapter provides a brief overview of TILLING (Targeting Induced Local Lesion in Genomes; a novel, reverse genetics approach that combines the advantages of point mutations provided by chemical mutagenesis with the advantages of PCR-based mutational screening) and Eco-TILLING [a method that uses TILLING techniques to look for natural mutations in individuals, usually for population genetics analysis] procedures, and the potential applications of both techniques in plants. Detailed descriptions of the conduct of TILLING and Eco-TILLING are provided.
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Resveratrols and flavonoids have important beneficial roles in plants and to humans. The grape family (Vitaceae) is one of several families that can biosynthesise both resveratrols and flavonoids. Previous research has shown that UV-C may induce the biosynthesis of resveratrols in the fruit of grape. But little is known about the UV-C-induced biosynthesis of resveratrols and flavonoids in grape leaves. This study demonstrated that the biosynthesis of resveratrol in grape leaves strongly increased in response to UV-C irradiation. The largest contribution to total resveratrols was trans-resveratrol. The expression of related genes, including PAL, C4H, 4CL and STS, increased and peaked 6–12 h after treatment, earlier than the peak of total resveratrol. In contrast, total flavonoid content and the expression of the key gene CHS was not affected by UV-C radiation, staying at a low level after UV-C irradiation. In summary, there is a differential response to UV-C radiation for the biosynthesis of stilbenes and flavonoids in grape leaves.
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Abstract Most of the genes of an organism are known from sequence, but most of the phenotypes are obscure. Thus, reverse genetics has become an important goal for many biologists. However, reverse-genetic methodologies are not similarly applicable to all organisms. In the general strategy for reverse genetics that we call TILLING (for Targeting Induced Local Lesions in Genomes), traditional chemical mutagenesis is followed by high-throughput screening for point mutations. TILLING promises to be generally applicable. Furthermore, because TILLING does not involve transgenic modifications, it is attractive not only for functional genomics but also for agricultural applications. Here, we present an overview of the status of TILLING methodology, including Ecotilling, which entails detection of natural variation. We describe public TILLING efforts in Arabidopsis and other organisms, including maize (Zea mays) and zebrafish. We conclude that TILLING, a technology developed in plants, is rapidly being adopted in other systems.
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Functional Genomics
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TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.
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