PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit

The high-throughput phenotypic analysis of Arabidopsis thaliana collections requires methodological progress and automation. Methods to impose stable and reproducible soil water deficits are presented and were used to analyse plant responses to water stress. Several potential complications and methodological difficulties were identified, including the spatial and temporal variability of micrometeorological conditions within a growth chamber, the difference in soil water depletion rates between accessions and the differences in developmental stage of accessions the same time after sowing. Solutions were found. Nine accessions were grown in four experiments in a rigorously controlled growth-chamber equipped with an automated system to control soil water content and take pictures of individual plants. One accession, An1, was unaffected by water deficit in terms of leaf number, leaf area, root growth and transpiration rate per unit leaf area. Methods developed here will help identify quantitative trait loci and genes involved in plant tolerance to water deficit.     

Plasticity to soil water deficit in Arabidopsis thaliana: dissection of leaf development into underlying growth dynamic and cellular variables reveals invisible phenotypes

Genetic variability in the plasticity of leaf area expansion in response to water deficit has been reported in Arabidopsis thaliana. Here, the objective was to identify the underlying dynamic and cellular processes involved in this variability. Twenty-five accessions were subjected to identical soil water deficit treatments. In all accessions, the plasticity of leaf production was low compared with that of individual leaf expansion. A subset of accessions was selected for further dissection of individual leaf expansion into its underlying variables: the rate and duration of leaf expansion and epidermal cell number and area. In all accessions, water deficit had opposite effects on the rate and duration of leaf expansion. The accumulation of these effects was reflected in changes in final leaf area. At the cellular level, moderate water deficits had opposite effects on cell number and cell size, but more severe ones reduced both variables. The importance of these opposing effects is highlighted by the behaviour of the accession An-1, for which the compensation between the decrease in leaf expansion rate and the increase in the duration of expansion is total. This dynamic plasticity in response to water deficit is not detectable when only final measurements are done.     

Integrated epigenome profiling of repressive histone modifications, DNA methylation and gene expression in normal and malignant urothelial cells

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20-30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5-10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer.     

Improved detection of bifidobacteria with optimised 16S rRNA-gene based pyrosequencing

The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, 'bifidobacteria-optimised' universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.     

Genetic affinities of inbred mouse strains of uncertain origin

Phylogenetic analyses of genetic data arising from 144 gene loci are used to describe the interrelationships among 24 widely used inbred strains of mice. An unordered-parsimony analysis gives a cladogram that is virtually identical to the known genealogy of the mouse strains. A loss-parsimony analysis is used to evaluate the hypothesis that the observed patterns of genetic divergence among these 24 strains can be explained by the segregation of residual heterozygosity arising from a small population of highly heterozygous mice. The loss-parsimony cladogram is very similar to both the unordered-parsimony cladogram and the known genealogy of the mice. The phylogenetic analyses of these 144 loci are integrated with data on the type and origin of the Y chromosome. Inclusion of the Y-chromosome data provides additional insights into the genetic composition of several of the original stocks used to produce the current inbred strains of mice. Ten strains of uncertain origin are contained in these analyses, including AKR, BUB, CE, I, NZB, P, RF, SJL, ST, and SWR. SJL is hypothesized to have been derived from the same Swiss albino stock previously used to produce SWR. The BUB strain appears to have had a complex origin and shows closest genetic similarity to SWR and ST. AKR and RF are shown to be closely related, while the I strain shows greatest genetic similarity to DBA/2 for the 144 loci. However, I and DBA possess different types of Y chromosome. The NZB strain shows genetic similarity to several stocks of both U.S. and European origins. The power of the genetic data used in these analyses reiterates that inbred strains of mice can be a valuable paradigm for studies in evolutionary biology.