Result of the Co-Response-Based Transcriptional Neighbourhood Search for 12 Ubiquitously Expressed Subtilase Genes

<p>The best 2% of positively correlated (A) and of negatively correlated (B) genes were selected and used to determine functional category representations. The upper chart represents the cluster tree resulting from our HCA analysis based on conversion of the enrichment of genes of particular functional categories into the Euclidean distances. In the lower chart, vertically stacked bar plots illustrate the distribution of functional categories of correlated gene for each of the 12 AtSBTs. For comparison, the functional category distribution of the genes represented in the underlying data matrices is shown to the left of each display.</p

Physical Map of AGI at TAIR Indicating the Chromosomal Distribution of the AtSBT Family

<p>The AtSBT genes are localized throughout the <i>Arabidopsis</i> genome as single genes or in tandem repeats.</p

Inferring Hypotheses on Functional Relationships of Genes: Analysis of the <em>Arabidopsis thaliana</em> Subtilase Gene Family

<div><p>The gene family of subtilisin-like serine proteases (subtilases) in <em>Arabidopsis thaliana</em> comprises 56 members, divided into six distinct subfamilies. Whereas the members of five subfamilies are similar to pyrolysins, two genes share stronger similarity to animal kexins. Mutant screens confirmed 144 T-DNA insertion lines with knockouts for 55 out of the 56 subtilases. Apart from SDD1, none of the confirmed homozygous mutants revealed any obvious visible phenotypic alteration during growth under standard conditions. Apart from this specific case, forward genetics gave us no hints about the function of the individual 54 non-characterized subtilase genes. Therefore, the main objective of our work was to overcome the shortcomings of the forward genetic approach and to infer alternative experimental approaches by using an integrative bioinformatics and biological approach. Computational analyses based on transcriptional co-expression and co-response pattern revealed at least two expression networks, suggesting that functional redundancy may exist among subtilases with limited similarity. Furthermore, two hubs were identified, which may be involved in signalling or may represent higher-order regulatory factors involved in responses to environmental cues. A particular enrichment of co-regulated genes with metabolic functions was observed for four subtilases possibly representing late responsive elements of environmental stress. The kexin homologs show stronger associations with genes of transcriptional regulation context. Based on the analyses presented here and in accordance with previously characterized subtilases, we propose three main functions of subtilases: involvement in (i) control of development, (ii) protein turnover, and (iii) action as downstream components of signalling cascades. Supplemental material is available in the Plant Subtilase Database (PSDB) (<a href="http://csbdb.mpimp-golm.mpg.de/psdb.html">http://csbdb.mpimp-golm.mpg.de/psdb.html</a>) , as well as from the CSB.DB (<a href="http://csbdb.mpimp-golm.mpg.de">http://csbdb.mpimp-golm.mpg.de</a>).</p></div

Bootstrapped Consensus Cluster Tree from Converted Detection Call Matrix of Affymetrix (Ath1) Microarray Experiments into Boolean Values (AtGenExpress Developmental Series)

<p>Cluster I covers ubiquitously expressed genes, whereas Cluster II mainly represents lowly or specifically expressed genes. These results were validated independently by semi-quantitative RT-PCR analysis (shown in the right panel). CL, cauline leaves; dS, dry seed; F, flower; R, root; RL, rosette leaves; Sd, seedling; Sq, siliques; St, inflorescence stem.</p

Bootstrapped Neighbour-Joining Tree Generated from an Alignment of the Predicted 56 AtSBT Full-Length Protein Sequences

<p>Groups of neighbouring genes (e.g., At3g46840 and At3g46850) are distinguished by specific colours.</p

Screenshot of the result page showing four identified peptides associated with the candidate hit protein which was tagged in the sample by 8 submitted fragment spectra

<p><b>Copyright information:</b></p><p>Taken from "ProMEX: a mass spectral reference database for proteins and protein phosphorylation sites"</p><p>http://www.biomedcentral.com/1471-2105/8/216</p><p>BMC Bioinformatics 2007;8():216-216.</p><p>Published online 23 Jun 2007</p><p>PMCID:PMC1920535.</p><p></p> For visual inspection of the spectra match, a north-south-plot of the library versus query spectra is shown below

Assessment of the effect of noise removal on the identification performance illustrated by the receiver operating characteristic (ROC)

<p><b>Copyright information:</b></p><p>Taken from "ProMEX: a mass spectral reference database for proteins and protein phosphorylation sites"</p><p>http://www.biomedcentral.com/1471-2105/8/216</p><p>BMC Bioinformatics 2007;8():216-216.</p><p>Published online 23 Jun 2007</p><p>PMCID:PMC1920535.</p><p></p> The identification rate is robust against different levels of peak noise filters up to 10%. We decided to use a conservative noise removal level of 2%. Recall (true positives) reflects the number of correctly identified peptides based on SEQUEST search, whereas 1 specificity corresponds to all ProMEX peptide identifications which did not match to the SEQUEST identifications. As a comparison, results from using the Euclidean distance are also included which proved to be inferior to the dot product

Heat-map visualization and cluster tree representations of amino acid contents and genotypes

Data were obtained from experiments where plants were starved of sulphate for 10 d. The heat-map was generated by using log base 2-transformed fold changes. The given data represent the ratio of the determined amino acids for control and starved plants. Each amino acid is represented by a single column and each genotype by a single row. Red indicates decreased relative metabolite content whereas blue indicates increased relative contents of amino acids compared with the wild-type. Separated heat-map visualization of amino acid contents in control and mutant plants are presented in in available at online and the respective diagrams in Fig. S2.<p><b>Copyright information:</b></p><p>Taken from "Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2831-2846.</p><p>Published online Jan 2008</p><p>PMCID:PMC2486478.</p><p></p

Contents of cysteine (upper row), γ-glutamylcysteine (GEC; middle row), and glutathione (GSH; lower row) are shown for plants overexpressing , , and , respectively, or down-regulated with respect to

Plants were grown for 10 weeks on soil before thiol extraction. knock-downs are represented by cross-hatched columns, overexpressing lines by white columns, and wild-type (WT) and empty-vector control lines (EV) by black columns. Values are the mean ±SD of three independent experiments. Asterisks indicate that the difference between the wild-type plants and the manipulated transgenic plants was significant using -tests ( ≤0.05).<p><b>Copyright information:</b></p><p>Taken from "Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2831-2846.</p><p>Published online Jan 2008</p><p>PMCID:PMC2486478.</p><p></p

Heat map generated from amino acid measurements reflecting log base 2-transformed and normalized amino acid levels and its similarity among themselves and the genotypes

The top colour bar indicates the relative log base 2-fold changes ranging between reduced relative (red) and increased relative (blue) contents of amino acids with respect to the wild-type.<p><b>Copyright information:</b></p><p>Taken from "Transcription factors relevant to auxin signalling coordinate broad-spectrum metabolic shifts including sulphur metabolism"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2831-2846.</p><p>Published online Jan 2008</p><p>PMCID:PMC2486478.</p><p></p