Dietary Restriction Depends on Nutrient Composition to Extend Chronological Lifespan in Budding Yeast Saccharomyces cerevisiae

10.1371/journal.pone.0064448PLoS ONE85

A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing

Background:We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates. Results:Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations. Conclusions:We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.Miriam Schreiber, Abdellah Barakate, Nicola Uzrek, Malcolm Macaulay, Adeline Sourdille, Jenny Morris, Pete E. Hedley, Luke Ramsay and Robbie Waug

Metabolomic analyses of Leishmania reveal multiple species differences and large differences in amino acid metabolism

Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts

Applications de la transgenèse à l'amélioration variétale du riz

Depuis l'obtention de la première plante transgénique de riz en 1988, de larges progrès ont été accomplis pour manipuler les caractères d'intérêt agronomique par ingénierie génétique chez cette plante d'importance alimentaire, économique et culturelle majeure. Les caractères concernés vont de la résistance aux insectes ravageurs et aux pathogènes fongiques, bactériens et viraux à la tolérance vis-à-vis des contraintes abiotiques (toxicités et carences minérales, température excessive ou basse, excès ou déficit en eau) en passant par les qualités nutritives ou industrielles du grain. Des portions entières de voies métaboliques peuvent à présent être finement manipulées, permettant par exemple la production de béta-carotène (provitamine A) dans l'albumen du grain, qui en est naturellement dépourvu, ou l'accumulation des enzymes de la photosynthèse C4 dans des tissus foliaires précis. La production de molécules d'intérêt thérapeutique ou industriel dans le grain a aussi été réalisée. Bien que certaines de ces innovations soient prêtes à être transférées dans le champ du riziculteur depuis plusieurs années, les politiques publiques sont encore hésitantes à franchir le pas. Ce retard relatif a été mis à profit pour conduire de nombreuses études sur les impacts alimentaires et environnementaux liés à la consommation et au déploiement de ces nouvelles cultures

MULTIPASS, a rice R2R3-type MYB transcription factor, regulates adaptive growth by integrating multiple hormonal pathways

Growth regulation is an important aspect of plant adaptation during environmental perturbations. Here, the role of MULTIPASS (OsMPS), an R2R3-type MYB transcription factor of rice, was explored. OsMPS is induced by salt stress and expressed in vegetative and reproductive tissues. Over-expression of OsMPS reduces growth under non-stress conditions, while knockdown plants display increased biomass. OsMPS expression is induced by abscisic acid and cytokinin, but is repressed by auxin, gibberellin and brassinolide. Growth retardation caused by OsMPS over-expression is partially restored by auxin application. Expression profiling revealed that OsMPS negatively regulates the expression of EXPANSIN (EXP) and cell-wall biosynthesis as well as phytohormone signaling genes. Furthermore, the expression of OsMPS-dependent genes is regulated by auxin, cytokinin and abscisic acid. Moreover, we show that OsMPS is a direct upstream regulator of OsEXPA4, OsEXPA8, OsEXPB2, OsEXPB3, OsEXPB6 and the endoglucanase genes OsGLU5 and OsGLU14. The multiple responses of OsMPS and its target genes to various hormones suggest an integrative function of OsMPS in the cross-talk between phytohormones and the environment to regulate adaptive growth. (Résumé d'auteur

La transformation génétique, un outil pour l'analyse fonctionnelle du génome du riz

Le riz ( Oryza sativa L.) a le double statut de céréale la plus consommée par l'homme et d'espèce modèle pour les monocotylédones. Élucider la fonction des gènes sous-tendant les principaux caractères d'intérêt agronomique chez cette plante contribuerait donc à accélérer non seulement l'amélioration variétale du riz mais aussi celle des autres céréales. La transformation génétique est un outil permettant d'accélérer la découverte de la fonction des gènes et l'innovation variétale. La première plante transgénique de riz a été obtenue dès 1988, et, depuis, les technologies ont régulièrement évolué vers plus d'efficacité et de précision. Nous présentons ici les grandes étapes de l'évolution de ces techniques, ainsi que leurs applications pour l'analyse fonctionnelle des gènes. En particulier, les perspectives ouvertes par de nouveaux sauts technologiques en cours, qui permettent à présent une modification ciblée des génomes sans introduction d'acide désoxyribonucléique (ADN) superflu, seront illustrées et discutées

Root cone angle is enlarged in docs1 LRR-RLK mutants in rice

Abstract Background The DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) gene belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) subfamily. It has been discovered few years ago in Oryza sativa (rice) in a screen to isolate mutants with defects in sensitivity to aluminum. The c68 (docs1–1) mutant possessed a nonsense mutation in the C-terminal part of the DOCS1 kinase domain. Findings We have generated a new loss-of-function mutation in the DOCS1 gene (docs1–2) using the CRISPR-Cas9 technology. This new loss-of-function mutant and docs1–1 present similar phenotypes suggesting the original docs1–1 was a null allele. Besides the aluminum sensitivity phenotype, both docs1 mutants shared also several root phenotypes described previously: less root hairs and mixed identities of the outer cell layers. Moreover, our new results suggest that DOCS1 could also play a role in root cap development. We hypothesized these docs1 root phenotypes may affect gravity responses. As expected, in seedlings, the early gravitropic response was delayed. Furthermore, at adult stage, the root gravitropic set angle of docs1 mutants was also affected since docs1 mutant plants displayed larger root cone angles. Conclusions All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development