1. The Power of Microarray Technology Ruth G. Alscher

2. Gene Expression: Control Points

3. Responses to Environmental Signals

4. Glycolysis, Citric Acid Cycle, and Related Metabolic Processes

5. Free Radicals Attack Cells: Survival Mechanisms?

6. ROS Arise as a Result of Exposure to: Ozone Sulfur dioxide High light Paraquat Extremes of temperature Salinity Drought

7. . Free radicals arise throughout the cell when stress is imposed

8. - Virginia Tech: determining which genes are essential for resistance to stress Plant Biologists: Drs. Alscher and Chevone., Cecilia Vasquez CS: Drs. Heath and Ramakrishnan, Margaret Ellis, Logan Hanks Statistics: Dr Key, Xiao Yang. NC State (Forest Biotechnology): discovering new genes in loblolly pine. Ying-Hsuan Sun, Drs. Sederoff and Whetten: Effects of Drought Stress on Gene Expression in Loblolly Pine Trees

9. Investigating gene expression patterns in stressed loblolly pine Selected cDNAs are spotted on to a glass surface (can be up to 20,000 different sequences spotted on to one slide). cDNAs derived from mRNA populations obtained from treated or control tree are hybridized to the cDNAs on the slide.

10. The Expression of Multiple Genes Can be Visualized Simultaneously Using Microarrays

11. Spots: (Sequences affixed to slide) 123 1 1 2 2 1 3 12 2 3 3 3 TreatmentControl Mix 123 Excitation Emission Detection Relative Abundance Detection Hybridization

12. The Expression of Multiple Genes Can be Visualized Simultaneously Using Microarrays

13. Detection of gene expression effects on microarrays Characterize gene function Test mutant phenotypes Genetic Regulatory Networks Identify mutants 1 2 3 4 Iterative strategy for detection of genetic interactions using microarrays

14. Clones on the drought-stress microarrays were replicated and randomly placed Experiment involved 384 archived pine ESTs Organized into 4 microtitre source plates after PCR Pipetted into 8 sets of 4 microtitre plates each Each set a different random arrangement of 384 ESTs Printed type A microarrays from first 4 sets Printed type B microarrays from second 4 sets Each array has 4 randomly placed replicates of each EST Each control versus stress comparison was done on 4 arrays — A and B; flip dyes; A and B Total of 16 replicates of each EST in each comparison Design of Microarrays

15. Who’s Who Ruth Alscher Plant Stress Boris Chevone Plant Stress Ron Sederoff, Ross Whetten Len van Zyl Y-H.Sun Forest Biotechnology Plant Biology Computer Science Lenwood Heath (CS) Algorithms Naren Ramakrishnan (CS) Data Mining Problem Solving Environments Craig Struble, Vincent Jouenne (CS) Image Analysis Statistics Ina Hoeschele (DS) Statistical Genetics Keying Ye (STAT) Bayesian Statistics Virginia Tech North Carolina State Univ. Virginia Tech Dawei Chen Molecular Biology Bioinformatics

16. Expresso People Ross Whetten Boris Chevone Ron Sederoff Y-H.SunDawei Chen Lenny Heath Ruth Alscher Vincent Jouenne Naren Ramakrishnan Keying Ye Len van Zyl Craig Struble

17. Hypotheses There is a group of genes whose expression confers resistance to drought stress. Expression of this group of genes is lower under severe than under mild stress. Individual members of gene families show distinct responses to drought stress.

18. Selection of cDNAs for Arrays 384 ESTs (xylem, shoot tip cDNAs of loblolly) were chosen on the basis of function and grouped into categories. Major emphasis was on processes known to be stress responsive. In cases where more than one EST had similar BLAST hits, all ESTs were used.

19. Integration of design and procedures Integration of image analysis tools and statistical analysis Connections to web databases and sequence alignment tools The software Aleph was used for inductive logic programming (ILP). Expresso: A Problem Solving Environment for Microarray Experiment Design and Analysis

20. Categories within Protective and Protected Processes Plant Growth Regulation Environmental Change Gene Expression Signal Transduction Protective Processes Protected Processes ROS and Stress Cell Wall Related Phenylpropanoid Pathway Development Metabolism Chloroplast Associated Carbon Metabolism Respiration and Nucleic Acids Mitochondrion Cells Tissues Cytoskeleton Secretion Trafficking Nucleus Protease-associated

21. A Note about Categories Categories are not mutually exclusive; gene(s) may be assigned to more then one category. For example, heat shock proteins have been grouped under these different categories and subcategories –Abiotic stress – heat –Gene expression – post-translational processing – chaperones –Abiotic stress - chaperones

22. Protective Processes Stress Cell Wall Related Phenylpropanoid Pathway Abiotic Biotic Antioxidant Processes Drought Heat Non-Plant Xenobiotics NADPH/Ascorbate/ Glutathione Scavenging Pathway Cytosolic ascorbate peroxidase Dehydrins, Aquaporins Heat shock proteins (Chaperones) superoxide dismutase-Fe superoxide dismutase-Cu-Zn glutathione reductase Sucrose Metabolism Cellulose Arabionogalactan proteins Hemicellulose Pectins Xylose Other Cell Wall Proteins isoflavone reductases phenylalanine ammonia-lyases S-adenosylmethionine decarboxylases glycine hydromethyltransferases Lignin Biosynthesis CCoAOMTs 4-coumarate-CoA ligases cinnamyl-alcohol dehydrogenase Chaperones “Isoflavone Reductases” GSTs Extensins and proline rich proteins Categories within “Protective Processes”

23. Quality Control Positive: LP-3, a loblolly gene known to respond positively to drought stress in loblloly pine, was included. LP-3 was positive in the moist versus mild comparison, and unchanged in the moist versus severe comparison. Negative: Four clones of human genes used as negative controls in the Arabidopsis Functional Genomics project were included. The clones did not respond.

24. Image Analysis: gridding, spot identification, intensity and background calculation, normalization Statistics: Fold or ratio estimation Combining replicates Higher-level Analysis: Clustering methods Inductive logic programming (ILP) Spot and Clone Analysis

25. Current Status of Expresso Completely automated and integrated –Statistical analysis –Data mining –Experiment capture in MEL Current Work: Integrating –Image processing –Querying by semi-structured views –Expresso-assisted experiment composition

26. Future Directions Next Generation Stress Chips 1.Time course, short and long term, to capture gene expression events underlying “emergency” and adaptive events following drought stress imposition. (Use all available ESTs for candidate stress resistance genes.) 2.Generate cDNA library from stressed seedlings. 3.Initiate modeling of kinetics of drought stress responses.

27. How to use microarrays to learn more about the influence of drought stress on gene expression? Where the biologists need the computer scientists. A. Confounding factors in the raw data 1. Limitations in accuracy (technique) 2. Biological variation (individuals) B. How to apply corrections for these confounding factors to maximize the predictive power of the data. C. Modeling regulatory networks. Microarray Data Analysis