Folate polyglutamylation is required for rice seed development

In plants, polyglutamylated folate forms account for a significant proportion of the total folate pool. Polyglutamylated folate forms are produced by the enzyme folylpolyglutamate synthetase (FPGS). The FPGS enzyme is encoded by two genes in rice, Os03g02030 and Os10g35940. Os03g02030 represents the major expressed form in developing seed. To determine the function of this FPGS gene in rice, a T-DNA knockout line was characterised. Disrupting Os03g02030 gene expression resulted in delayed seed filling. LC-MS/MS-based metabolite profiling revealed that the abundance of mono- and polyglutamylated folate forms was significantly decreased in seeds of the knockout line. RT-qPCR detected an increase in the transcript abundance of folate biosynthesis genes in seed of the knockout plant, whereas the folate deglutamating enzyme ?-glutamyl hydrolase mRNA level was reduced. Our study has uncovered a novel role for folate polyglutamylation during rice seed development and a potential feedback mechanism to maintain folate abundance.(Résumé d'auteur

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

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

10.1371/journal.pone.0064448PLoS ONE85

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

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

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

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