PathExpress: a web-based tool to identify relevant pathways in gene expression data

PathExpress is a web-based tool developed to interpret gene expression data obtained from microarray experiments by identifying the most relevant metabolic pathways associated with a subset of genes (e.g. differentially expressed genes). A graphical pathway representation permits the visualization of the expressed genes in a functional context. Based on the publicly accessible KEGG Ligand database, PathExpress can be adapted to any organism and is currently available for seven Affymetrix genome arrays. About 20% of the probe sets of each array have been assigned to Enzyme Commission numbers by homology relationship and linked to corresponding metabolic pathways

Genome-wide transcriptional analysis of super-embryogenic Medicago truncatula explant cultures

BACKGROUND: The Medicago truncatula (M. truncatula) line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than its wild type progenitor Jemalong. To understand the molecular basis for the regeneration capacity of this super-embryogenic line 2HA, using Affymetrix GeneChip®, we have compared transcriptomes of explant leaf cultures of these two lines that were grown on media containing the auxin NAA (1-naphthaleneacetic acid) and the cytokinin BAP (6-benzylaminopurine) for two weeks, an early time point for tissue culture proliferation. RESULTS: Using Affymetrix GeneChip®, GCRMA normalisation and statistical analysis, we have shown that more than 196 and 49 probe sets were significantly (p < 0.05) up- or down-regulated respectively more than 2 fold in expression. We have utilised GeneBins, a database for classifying gene expression data to distinguish differentially displayed pathways among these two cultures which showed changes in number of biochemical pathways including carbon and flavonoid biosynthesis, phytohormone biosynthesis and signalling. The up-regulated genes in the embryogenic 2HA culture included nodulins, transporters, regulatory genes, embryogenesis related arabinogalactans and genes involved in redox homeostasis, the transition from vegetative growth to reproductive growth and cytokinin signalling. Down-regulated genes included protease inhibitors, wound-induced proteins, and genes involved in biosynthesis and signalling of phytohormones auxin, gibberellin and ethylene. These changes indicate essential differences between the super-embryogenic line 2HA and Jemalong not only in many aspects of biochemical pathways but also in their response to auxin and cytokinin. To validate the GeneChip results, we used quantitative real-time RT-PCR to examine the expression of the genes up-regulated in 2HA such as transposase, RNA-directed DNA polymerase, glycoside hydrolase, RESPONSE REGULATOR 10, AGAMOUS-LIKE 20, flower promoting factor 1, nodulin 3, fasciclin and lipoxygenase, and a down-regulated gene ETHYLENE INSENSITIVE 3, all of which positively correlated with the microarray data. CONCLUSION: We have described the differences in transcriptomes between the M. truncatula super-embryogenic line 2HA and its non-embryogenic progenitor Jemalong at an early time point. This data will facilitate the mapping of regulatory and metabolic networks involved in the gaining totipotency and regeneration capacity in M. truncatula and provides candidate genes for functional analysis

Genome-wide transcriptional analysis of super-embryogenic Medicago truncatula explant cultures

Background. The Medicago truncatula (M. truncatula) line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than its wild type progenitor Jemalong. To understand the molecular basis for the regeneration capacity of this super-embryogenic line 2HA, using Affymetrix GeneChip, we have compared transcriptomes of explant leaf cultures of these two lines that were grown on media containing the auxin NAA (1-naphthaleneacetic acid) and the cytokinin BAP (6-benzylaminopurine) for two weeks, an early time point for tissue culture proliferation. Results. Using Affymetrix GeneChip, GCRMA normalisation and statistical analysis, we have shown that more than 196 and 49 probe sets were significantly (p < 0.05) up- or down-regulated respectively more than 2 fold in expression. We have utilised GeneBins, a database for classifying gene expression data to distinguish differentially displayed pathways among these two cultures which showed changes in number of biochemical pathways including carbon and flavonoid biosynthesis, phytohormone biosynthesis and signalling. The up-regulated genes in the embryogenic 2HA culture included nodulins, transporters, regulatory genes, embryogenesis related arabinogalactans and genes involved in redox homeostasis, the transition from vegetative growth to reproductive growth and cytokinin signalling. Down-regulated genes included protease inhibitors, wound-induced proteins, and genes involved in biosynthesis and signalling of phytohormones auxin, gibberellin and ethylene. These changes indicate essential differences between the super-embryogenic line 2HA and Jemalong not only in many aspects of biochemical pathways but also in their response to auxin and cytokinin. To validate the GeneChip results, we used quantitative real-time RT-PCR to examine the expression of the genes up-regulated in 2HA such as transposase, RNA-directed DNA polymerase, glycoside hydrolase, RESPONSE REGULATOR 10, AGAMOUS-LIKE 20, flower promoting factor 1, nodulin 3, fasciclin and lipoxygenase, and a down-regulated gene ETHYLENE INSENSITIVE 3, all of which positively correlated with the microarray data. Conclusion. We have described the differences in transcriptomes between the M. truncatula super-embryogenic line 2HA and its non-embryogenic progenitor Jemalong at an early time point. This data will facilitate the mapping of regulatory and metabolic networks involved in the gaining totipotency and regeneration capacity in M. truncatula and provides candidate genes for functional analysis

The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive

BACKGROUND The Medicago truncatula 2HA seed line is highly embryogenic while the parental line Jemalong rarely produces embryos. The 2HA line was developed from one of the rare Jemalong regenerates and this method for obtaining a highly regenerable genotype in M. truncatula is readily reproducible suggesting an epigenetic mechanism. Microarray transcriptomic analysis showed down regulation of an ETHYLENE INSENSITIVE 3-like gene in 2HA callus which provided an approach to investigating epigenetic regulation of genes related to ethylene signalling and the 2HA phenotype. Ethylene is involved in many developmental processes including somatic embryogenesis (SE) and is associated with stress responses. RESULTS Microarray transcriptomic analysis showed a significant number of up-regulated transcripts in 2HA tissue culture, including nodule and embryo specific genes and transposon-like genes, while only a few genes were down-regulated, including an EIN3-like gene we called MtEIL1. This reduced expression was associated with ethylene insensitivity of 2HA plants that was further investigated. The weak ethylene insensitivity affected root and nodule development. Sequencing of MtEIL1 found no difference between 2HA and wild-type plants. DNA methylation analysis of MtEIL1 revealed significant difference between 2HA and wild-type plants. Tiling arrays demonstrated an elevated level of miRNA in 2HA plants that hybridised to the antisense strand of the MtEIL1 gene. AFLP-like methylation profiling revealed more differences in DNA methylation between 2HA and wild-type. Segregation analysis demonstrated the recessive nature of the eil1 phenotype and the dominant nature of the SE trait. CONCLUSIONS We have demonstrated that EIL1 of Medicago truncatula (MtEIL1) is epigenetically silenced in the 2HA seed line. The possible cause is an elevated level of miRNA that targets its 3'UTR and is also associated with DNA methylation of MtEIL1. Down regulation of MtEIL1 makes it possible to form nodules in the presence of ethylene and affects root growth under normal conditions. Segregation analysis showed no association between MtEIL1 expression and SE in culture but the role and mechanism of ethylene signalling in the process of plant regeneration through SE requires further investigation. The work also suggests that epigenetic changes to a particular gene induced in culture can be fixed in regenerated plants.This work was funded by the Australian Research Council (CEO348212) through the ARC Centre of Excellence for Integrative Legume Research (CILR)

Transcriptional profiling of Medicago truncatula meristematic root cells

BACKGROUND: The root apical meristem of crop and model legume Medicago truncatula is a significantly different stem cell system to that of the widely studied model plant species Arabidopsis thaliana. In this study we used the Affymetrix Medicago GeneChip® to compare the transcriptomes of meristem and non-meristematic root to identify root meristem specific candidate genes. RESULTS: Using mRNA from root meristem and non-meristem we were able to identify 324 and 363 transcripts differentially expressed from the two regions. With bioinformatics tools developed to functionally annotate the Medicago genome array we could identify significant changes in metabolism, signalling and the differentially expression of 55 transcription factors in meristematic and non-meristematic roots. CONCLUSION: This is the first comprehensive analysis of M. truncatula root meristem cells using this genome array. This data will facilitate the mapping of regulatory and metabolic networks involved in the open root meristem of M. truncatula and provides candidates for functional analysis.This research is supported by a grant from the Australian Research Council Centre of Excellence Program (CE0348212). PH was supported by an Australian Postgraduate Award. We thank Lily Shen from the ANU Electron Microscopy Unit for assistance with microscopy

Transcriptional profiling of Medicago truncatula meristematic root cells

<p>Abstract</p> <p>Background</p> <p>The root apical meristem of crop and model legume <it>Medicago truncatula </it>is a significantly different stem cell system to that of the widely studied model plant species <it>Arabidopsis thaliana</it>. In this study we used the Affymetrix <it>Medicago </it>GeneChip^®to compare the transcriptomes of meristem and non-meristematic root to identify root meristem specific candidate genes.</p> <p>Results</p> <p>Using mRNA from root meristem and non-meristem we were able to identify 324 and 363 transcripts differentially expressed from the two regions. With bioinformatics tools developed to functionally annotate the <it>Medicago </it>genome array we could identify significant changes in metabolism, signalling and the differentially expression of 55 transcription factors in meristematic and non-meristematic roots.</p> <p>Conclusion</p> <p>This is the first comprehensive analysis of <it>M. truncatula </it>root meristem cells using this genome array. This data will facilitate the mapping of regulatory and metabolic networks involved in the open root meristem of <it>M. truncatula </it>and provides candidates for functional analysis.</p

Bioinformatic analysis of the CLE signaling peptide family

Background. Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases. Results. We identified 114 new members of the CLE protein family from various plant species, as well as five protein sequences containing multiple CLE domains. We were able to cluster the CLE domain proteins into 13 distinct groups based on their pairwise similarities in the primary CLE motif. In addition, we identified secondary motifs that coincide with our sequence clusters. The groupings based on the CLE motifs correlate with known biological functions of CLE signaling peptides and are analogous to groupings based on phylogenetic analysis and ectopic overexpression studies. We tested the biological function of two of the predicted CLE signaling peptides in the legume Medicago truncatula. These peptides inhibit the activity of the root apical and lateral root meristems in a manner consistent with our functional predictions based on other CLE signaling peptides clustering in the same groups. Conclusion. Our analysis provides an identification and classification of a large number of novel potential CLE signaling peptides. The additional motifs we found could lead to future discovery of recognition sites for processing peptidases as well as predictions for receptor binding specificity

GeneBins: a database for classifying gene expression data, with application to plant genome arrays

BACKGROUND: To interpret microarray experiments, several ontological analysis tools have been developed. However, current tools are limited to specific organisms. RESULTS: We developed a bioinformatics system to assign the probe set sequences of any organism to a hierarchical functional classification modelled on KEGG ontology. The GeneBins database currently supports the functional classification of expression data from four Affymetrix arrays; Arabidopsis thaliana, Oryza sativa, Glycine max and Medicago truncatula. An online analysis tool to identify relevant functions is also provided. CONCLUSION: GeneBins provides resources to interpret gene expression results from microarray experiments. It is available a

Proteome Analysis of Pod and Seed Development in the Model Legume Lotus japonicus

Legume pods serve important functions during seed development and are themselves sources of food and feed. Compared to seeds, the metabolism and development of pods are not well-defined. The present characterization of pods from the model legume Lotus japonicus, together with the detailed analyses of the pod and seed proteomes in five developmental stages, paves the way for comparative pathway analysis and provides new metabolic information. Proteins were analyzed by two-dimensional gel electrophoresis and tandem-mass spectrometry. These analyses lead to the identification of 604 pod proteins and 965 seed proteins, including 263 proteins distinguishing the pod. The complete data set is publicly available at http://www.cbs.dtu.dk/cgi-bin/lotus/db.cgi, where spots in a reference map are linked to experimental data, such as matched peptides, quantification values, and gene accessions. Identified pod proteins represented enzymes from 85 different metabolic pathways, including storage globulins and a late embryogenesis abundant protein. In contrast to seed maturation, pod maturation was associated with decreasing total protein content, especially proteins involved in protein biosynthesis and photosynthesis. Proteins detected only in pods included three enzymes participating in the urea cycle and four in nitrogen and amino group metabolism, highlighting the importance of nitrogen metabolism during pod development. Additionally, five legume seed proteins previously unassigned in the glutamate metabolism pathway were identified

Cent scientifiques répliquent à SEA (Suppression des Expériences sur l’Animal vivant) et dénoncent sa désinformation

La lutte contre la maltraitance animale est sans conteste une cause moralement juste. Mais elle ne justifie en rien la désinformation à laquelle certaines associations qui s’en réclament ont recours pour remettre en question l’usage de l’expérimentation animale en recherche