PII: S1360-1385(99)01510-1

T he wild crucifer Arabidopsis thaliana has become an important model system because it allows genetics to combine with molecular biology (i.e. it has a short generation time, a small genome and is easily transformed) Genetic variation is required for the functional analysis of the Arabidopsis genome Laboratory-induced mutants Currently, the functional analysis of Arabidopsis genes and the dissection of complex traits are based largely on the phenotypic characterization of mutants selected by forward and reverse genetics in a few laboratory 'wild-type' genotypes. The inbred strains generally used are Landsberg erecta (Ler), Columbia (Col) and Wassilewskija (Ws), originally collected from the wild by the pioneers of Arabidopsis research, Friedrich Laibach and colleagues 3 . These forward and reverse genetic approaches using classical (physical or chemical) and insertional (biological) mutagens have proven their usefulness 1 . The possibility of identifying genotypes with an insertion in a gene of known nucleotide sequence, independently of the presence of a phenotype, has led to large-scale projects for disrupting most of the Arabidopsis genes Naturally occurring variants: new times for an old resource As an alternative to generating laboratory-induced mutants, another source of genetic variation can be found among and within naturally occurring populations of Arabidopsis Exploitation of the genetic variation among accessions has been limited because of its mostly quantitative (continuous) nature, in contrast with the commonly studied mutants, which provide qualitative (discrete) variation. This dichotomy is defined basically by the number of loci and the environmental effect underlying the variation under study, which determine the tools used for its analysis. Only in the past decade, with the advent of efficient molecular marker technologies and specific statistical methods, has the map position and the effects of quantitative trait loci (QTL) been establishe

Three-color dSTORM Imaging and Analysis of Recombination Foci in Mouse Spread Meiotic Nuclei

During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience.</p

Integrative analyses of genetic variation in enzyme activities of primary carbohydrate metabolism reveal distinct modes of regulation in Arabidopsis thaliana

Multiparallel QTL analysis of 15 Arabidopsis primary carbohydrate metabolism enzymes reveals that traits affecting primary metabolism are often correlated

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to 'rescue' a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.

HEATSTORE: high temperature underground thermal energy storage

Thermal energy storage technologies need to be further developed and need to become an integral component in the future energy system infrastructure to meet variations in both the availability and demand of energy. The main objectives of project HEATSTORE are to lower the cost, reduce risks, improve the performance of high temperature (~25°C to ~90°C) underground thermal energy storage (HT-UTES) technologies and to optimize heat network demand side management (DSM). This is primarily achieved by 6 new demonstration pilots and 8 case studies of existing systems with distinct configurations of heat sources, heat storage and heat utilization. It will advance the commercial viability of HT-UTES technologies and, through an optimized balance between supply, transport, storage and demand, enable geothermal energy production to reach its maximum deployment potential in the European energy transition. HEATSTORE is a project under the GEOTHERMICA – ERA NET Cofund and contributes to achieving the several objectives of accelerating the uptake of geothermal energy by 1) advancing and integrating different types of underground thermal energy storage (UTES) in the energy system, 2) providing a means to maximize geothermal heat production and optimize the business case of geothermal heat production doublets, 3) addressing technical, economic, market, environmental, regulatory and policy aspects that are necessary to support efficient and cost-effective deployment of UTES technologies in Europe. The 3-year project started in 2018 will stimulate a fast-track market uptake in Europe, promoting development from demonstration phase to commercial deployment within 2 to 5 years, and provide an outlook for utilization potential towards 2030 and 2050. The HEATSTORE consortium brings together 23 contributing partners (mix of scientific research institutes and private companies) from 9 countries.HEATSTORE (170153-4401) is one of nine projects under the GEOTHERMICA – ERA NET Cofund aimed at accelerating the uptake of geothermal energy. The GEOTHERMICA project is supported by the European Union’s HORIZON 2020 programme for research, technological development and demonstration under grant agreement No 731117.Postprint (published version

Co-Variation between Seed Dormancy, Growth Rate and Flowering Time Changes with Latitude in Arabidopsis thaliana

Life-history traits controlling the duration and timing of developmental phases in the life cycle jointly determine fitness. Therefore, life-history traits studied in isolation provide an incomplete view on the relevance of life-cycle variation for adaptation. In this study, we examine genetic variation in traits covering the major life history events of the annual species Arabidopsis thaliana: seed dormancy, vegetative growth rate and flowering time. In a sample of 112 genotypes collected throughout the European range of the species, both seed dormancy and flowering time follow a latitudinal gradient independent of the major population structure gradient. This finding confirms previous studies reporting the adaptive evolution of these two traits. Here, however, we further analyze patterns of co-variation among traits. We observe that co-variation between primary dormancy, vegetative growth rate and flowering time also follows a latitudinal cline. At higher latitudes, vegetative growth rate is positively correlated with primary dormancy and negatively with flowering time. In the South, this trend disappears. Patterns of trait co-variation change, presumably because major environmental gradients shift with latitude. This pattern appears unrelated to population structure, suggesting that changes in the coordinated evolution of major life history traits is adaptive. Our data suggest that A. thaliana provides a good model for the evolution of trade-offs and their genetic basis.<br

Phenotype of Arabidopsis thaliana semi-dwarfs with deep roots and high growth rates under water-limiting conditions is independent of the GA5 loss-of-function alleles

BACKGROUND AND AIMS: The occurrence of Arabidopsis thaliana semi-dwarf accessions carrying inactive alleles at the gibberellin (GA) biosynthesis GA5 locus has raised the question whether there are pleiotropic effects on other traits at the root level, such as rooting depth. In addition, it is unknown whether semi-dwarfism in arabidopsis confers a growth advantage under water-limiting conditions compared with wild-type plants. The aim of this research was therefore to investigate whether semi-dwarfism has a pleiotropic effect in the root system and also whether semi-dwarfs might be more tolerant of water-limiting conditions. METHODS: The root systems of different arabidopsis semi-dwarfs and GA biosynthesis mutants were phenotyped in vitro using the GROWSCREEN-ROOT image-based software. Semi-dwarfs were phenotyped together with tall, near-related accessions. In addition, root phenotypes were investigated in soil-filled rhizotrons. Rosette growth trajectories were analysed with the GROWSCREEN-FLUORO setup based on non-invasive imaging. KEY RESULTS: Mutations in the early steps of the GA biosynthesis pathway led to a reduction in shoot as well as root size. Depending on the genetic background, mutations at the GA5 locus yielded phenotypes characterized by decreased root length in comparison with related wild-type ones. The semi-dwarf accession Pak-3 showed the deepest root system both in vitro and in soil cultivation experiments; this comparatively deep root system, however, was independent of the ga5 loss-of-function allele, as shown by co-segregation analysis. When the accessions were grown under water-limiting conditions, semi-dwarf accessions with high growth rates were identified. CONCLUSIONS: The observed diversity in root system growth and architecture occurs independently of semi-dwarf phenotypes, and is probably linked to a genetic background effect. The results show that there are no clear advantages of semi-dwarfism at low water availability in arabidopsis.International Max Planck Research School PhD Fellowship/[]/IMPRS/AlemaniaHelmholtz Association//[]//AlemaniaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro para Investigaciones en Granos y Semillas (CIGRAS