1. “My Experiment” and What I Want to Discover My experiment involved comparing the effect of long term ozone exposure on gene expression in a wild type and a mutant genotype (only one gene, Gene G, difference). Time points taken. What effect does the mutated gene have on defense against ozone? The two genotypes showed a phenotypic difference under ozone, e.g. the mutant genotype senesced sooner (got old) than the wild type. Phenotype can be displayed as numbers on a time scale. I want to identify any pathways, or fragments thereof, and their corresponding (hypothetical or proven) regulators, that behaved differently between the two genotypes, starting with lists of genes. I want to be able to capture an image of that pathway fragment, with the indication that ozone affected its function.

2. Reported Actions of Ozone Gene G Senescence onset is a quantifiable phenotype, which is the consequence of responses of processes and entities named in the boxes below,( transcription etc…). Modified from Ludwikow A, Sadowski J. J Integr Plant Biol. 2008 Oct;50(10):1256-67

3. Desiderata for Inference: A Fancy Output Doc Anti-Aging for Plants. Access MapMan, PlantMetGen Map, to get information on enriched metabolic and signaling pathways that are differentially affected by ozone in the two genotypes. Images captured from the first two, with data marked. Mine enriched GO terms/MapMan bins to search for possible regulators that may have responded to ozone, e,g. possible components of signaling pathways such as transcription factors/ protein kinases, or other regulatory functions such as modifiers of chromatin structure, or small RNAs. For example, putative Regulator X did not respond to ozone in the mutant genotype. Regulator X and Pathway A were activated in the wild type under ozone, and were unresponsive in the mutant genotype. Infer that the Gene G that was mutated controls Regulator X which is essential for defense against ozone, by protecting against early senescence (directly or indirectly). Ability to draw, literally, draw, this inference. Yes, Greg, these are correlations!.

4. Drawing the Inference The connection from Gene G to the AND above Regulator R is the inferred causality link. One Interpretation: Pathway A operates to control the time of senescence onset. The change in onset of senescence may be exerted through the hormones that respond to ozone. Phenotype Perturbation

5. Methods of Causal Inference * Granger Causality: Uses statistical correlation to assess causality between two time courses Bayesian Networks: A network of conditional probabilities used to build model; tested statistically Structured Equation Modeling: A network of functional dependencies used to build model; tested statistically *Thanks to Björn for these.

6. Luck, or the Lack Thereof If we are very lucky, only one pathway, and one putative regulator, will be identified as being differently affected by ozone in the two genotypes. We could then draw a simple genotype to phenotype inference. A much more likely result are data pointing to a number of interlocking pathways. We could draw that too!

7. Hormones and Ozone Modified from Ludwikow A, Sadowski J. J Integr Plant Biol. 2008 Oct;50(10):1256- 67 When ozone is present, four plant hormones respond, which interact with each other. 1 1 2 2 3 3 4 4

8. Another Version of Ozone Action Wrzaczek * et al. BMC Plant Biology 2010, 10:95 Interlocking pathways abound!

9. GUIGUI Design info/file GEO# GOterm of interest RNAseq data* Metabolomics data* Proteomics data* GenExpr2Ddata GenExprSum ContrastFiles EnrichedGO graphs GeneMANIA output G2P inferences AffyGen Analyser ® AffyGen Analyser ® Cytoscape BiNGO Enriched GO terms/Bins/ Pathways Enriched GO terms/Bins/ Pathways GeneMANIA ** Gene List(s) iPlantOmicsVis --Analysis & Visualization of Gene Expression, Proteomics and Metabolite Data Plant MetGenMAP User MapMen GO® Analyser Ondex** Output docs * statistically analyzed data Statistically Analyzed data* ViVA eFP ** ** tools that provide more information/ connections from the literature Phenotypic observations* feed into ViVa/ output docs Effect of Environmental Change on Species/Genotypes, Developmental Time Course in Species/Genotypes