1. El PMN: Tu amigo en el metabolismo de plantas Kate Dreher curator PMN/AraCyc/TAIR

2. PMN = The Plant Metabolic Network Created in 2008 Funded by the National Science Foundation www. plantcyc.org How do I find information? How do I build a metabolic pathway database for my favorite plant? Putting the PMN to use Sue Rhee (PI) Peifen Zhang (Director)

3. Choose a database... Use the basic search tools Finding the information you want...

4. Basic searching: practice problems 1. What are some of the pathways that talk about salt stress in the PMN? 2. How many amides are in PlantCyc? 3. Are there more PlantCyc or AraCyc reactions listed under the E.C. category 3 (hydrolases)?

5. Basic searching: practice problems 1. What are some of the pathways that talk about salt stress in the PMN? Arabidopsis thaliana proline biosynthesis I Measurement of free proline content and gene expression and enzyme activity levels from salt-stress treated plants [ Roosens98 ] showed that in young... Arabidopsis thaliana choline biosynthesis I Arabidopsis thaliana choline biosynthesis I Summers93: Summers PS, Weretilnyk EA (1993). "Choline Synthesis in Spinach in Relation to Salt Stress." Plant Physiol 103(4);1269-1276. PMID: 12232019... Arabidopsis thaliana arginine biosynthesis III Arabidopsis thaliana arginine biosynthesis III "Isolation of the ornithine-delta-aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana." Plant Physiol 117(1);263-71.... PlantCyc phosphatidylcholine biosynthesis I PlantCyc phosphatidylcholine biosynthesis I "Regulation of phosphatidylcholine biosynthesis under salt stress involves choline kinases in Arabidopsis thaliana." FEBS Lett 566(1-3);115-20.... PlantCyc putrescine biosynthesis via N-carbamoylputrescine PlantCyc putrescine biosynthesis via N-carbamoylputrescine... and biosynthetic gene expression in Arabidopsis thaliana under salt stress.... knockout mutation of ADC2 gene reveals inducibility by osmotic stress.... 2. How many amides are in PlantCyc? 9 3. Are there more PlantCyc or AraCyc reactions listed under the E.C. category 3 (hydrolases)? More AraCyc reactions

6. Basic searching: practice problems Find a pathway in PlantCyc whose end product is tryptophan. Is it part of any superpathways? How many anthinilate synthases from Medicago truncatula are associated with this pathway? Is there experimental or computational support for their assignment to this reaction? Which enzyme is subject to feedback regulation? Please give gene code, too. What type of experimental support exists for this enzyme? What is its Km for chorismate? Find the protein sequence for the closest ortholog in papaya What is the length of the full-length coding sequence for its closest homolog in soybean (in soybeans)? How many species are associated with reaction 5.3.1.24? Find a paper that talks about an enzyme with similar activity in Zea mays? What is the chemical formula of anthranilate? What are two synonyms for this compound? What other pathway is it found in?

7. Basic information: practice problems Find a pathway in PlantCyc whose end product is tryptophan. TRPSYN-PWY – tryptophan biosynthesis Is it part of any superpathways? YES, superpathway of phenylalanine, tyrosine and tryptophan biosynthesis superpathway of phenylalanine, tyrosine and tryptophan biosynthesis How many anthinilate synthases from Medicago truncatula are associated with this pathway? 4 Is there experimental or computational support for their assignment to this reaction? Computational Which enzyme is subject to feedback regulation? anthranilate synthase, At5g05730 What type of experimental support exists for this enzyme? mutant phenotype; genetic interaction What is its Km for chorismate? 180 uM Find the protein sequence for the closest ortholog in papaya MQTLGFSYRLVPSGRRFSPVPANGISGARSSSLVNVRTFKCMSLSSPSLVCDVKKFAEASKHGNVVPLYH SIFSDQLTPVLAYRCLVKEDDREAPSFLFESVEPGFQASSVGRYSVVGAQPTIEIVAKENKVTIMDHEGG TLSEEYVQDPMMIPRRISEGWKPQLIDELPDTFCGGWVGYFSYDTIRYVEKKKLPFSMAPEDDRNLADIH LGLYDDVIVFDHVEKKAHVIHWVRLDQYSSAEKAYNDGLKRLEKLVAKVQDIDPPRLSPGSVDLQTRQFG PSLRKSTMTSEEYKMAVLEAKEHILAGDIFQIVLSQRFERRTFADPFEVYRALRVVNPSPYMTYLQARGC ILVASSPEILTRVEKKKIVNRPLAGTVRRGKTTAEDEMLEKQLLNDAKQCAEHIMLVDLGRNDVGKVTGE LHDHLTCWDVLRAALPVGTVSGAPKVKAMELIDQLEVTRRGPYSGGFGGISFTGNMDVALALRTIVFPTG THYNTMYSYKDVENRRDWIAHLQAGAGIVADSNPDDENQECHNKVAGLARAIDLAESAFVNK* What is the length of the full-length coding sequence for its closest homolog in soybean? 1593 bp How many species are associated with reaction 5.3.1.24? three Find a paper that talks about an enzyme with similar activity in Zea mays? Some physical characteristics of the enzymes of L-tryptophan biosynthesis in higher plants What is the chemical formula of anthranilate? C7H7NO2 What are two synonyms for this compound? anthranilic acid, 2-aminobenzoic acid What other pathway is it found in? acridone alkaloid biosynthesis

8. Advanced searching Advanced Query Form Downloading the whole database

9. Advanced searching: practice problems Find a list of all of the enzymes with a pI between 5 and 6.5 that are glucosyltransferases. What species are they found in? What is the highest pI and the lowest pI in the group? Get a list of all of the reactions that are children of E.C. 2.4.1. and identify the compounds in the right and the left How many reactions are in your original list? If you eliminate all of the reactions that have rhamnose in their name, how many do you have left? Look for all the pathways that have more than one hypothetical reaction in them. Get all of the citations associated with them How many of the pathways have more than 4 hypothetical reactions associated with them?

10. Advanced searching: practice problems Find a list of all of the enzymes in PlantCyc with a pI between 5 and 6.5 that are glucosyltransferases. How many are there? What species are they found in? What is the highest pI and the lowest pI in the group? Get a list of all of the reactions that are children of E.C. 2.4.1. in PlantCyc and identify the compounds in the right and the left in your output list How many reactions are in your original list? If you eliminate all of the reactions that have rhamnose in their name, how many do you have left? Look for all the pathways in PlantCyc that have more than one hypothetical reaction in them. Get all of the citations associated with them How many of the pathways have more than 4 hypothetical reactions associated with them?

11. Advanced searching: practice problems Find a list of all of the enzymes in PlantCyc with a pI between 5 and 6.5 that are glucosyltransferases. How many are there? 13 What species are they found in? Zea mays, Dorotheanthus bellidiformis, Cicer arietinum, Brassica napus Zea maysDorotheanthus bellidiformisCicer arietinumBrassica napus Pinus strobus What is the highest pI and the lowest pI in the group? 5, 6.36 Get a list of all of the reactions that are children of E.C. 2.4.1. in PlantCyc and identify the compounds in the right and the left in your output list How many reactions are in your original list? 189 If you eliminate all of the reactions that have rhamnose in their name, how many do you have left? 174 Look for all the pathways in PlantCyc that have more than one hypothetical reaction in them. 17 Get all of the citations associated with them How many of the pathways have more than 4 hypothetical reactions associated with them? 6

12. OMICs viewer Do your experiment (or get your data) Create your input file Upload and analyze your data Absolute values Time course Relative values

13. OMICs viewer: practice problems Use PMN_taller_OMICS_practice_problem The columns are: 0 – gene identifiers 1 – time point 1 2 – time point 2 3 – time point 3 4 – rapid change (ratio of time point 1 to 2) Find the pathways that have gene expression values of over 600 at time point 2. Which one has the highest number of steps affected in the pathway? Create an animation of all three time points Look for pathways that had genes whose expression changed more than 4-fold between the first and last time points. How many appear? Use the data you have already calculated and look for pathways that have gene expression changes of more than 6-fold in the first time interval. How many appear?

14. OMICs viewer: practice problems Use PMN_taller_OMICS_practice_problem The columns are: 0 – gene identifiers 1 – time point 1 2 – time point 2 3 – time point 3 4 – rapid change (ratio of time point 1 to 2) Find the pathways that have gene expression values of over 600 at time point 2. Which one has the highest number of steps affected in the pathway? starch degradation Create an animation of all three time points Look for pathways that had genes whose expression changed more than 4-fold between the first and last time points. How many appear? 13 Use the data you have already calculated and look for pathways that have gene expression changes of more than 6-fold in the first time interval. How many appear? 6

15. Building your own metabolic database Step 1: Genes / Nucleotide Sequences Genome sequencing Unigene builds ESTs Step 2: Annotate Predicted Proteins Annotation source: PMN pipeline JGI or other sequencing consortium Small group of dedicated scientists, etc. Annotation tasks: Give enzymes a name Add GO molecular function terms Assign to a MetaCyc or EC reaction Use Pathologic and MetaCyc or PlantCyc to create new pathway database Remove non-enzymatic proteins before prediction After first round of prediction, review unassigned enzymes Repredict Validate new database

16. Validating your own metabolic database PathoLogic errs on the side of over-prediction Curators / Scientists validate pathways...

17. Examine predicted pathways Search for evidence in published papers, books, etc. Is the pathway described in the literature for my species? Are the crucial metabolites described in in the literature for my species? Are there unique reactions associated with the pathway that have assigned genes? Is there evidence that this is a universal plant pathway? Is it on the PMN green list? Is there evidence that this pathway is NEVER found in plants? Only relevant when prediction is made using MetaCyc Is it on the PMN black list?

18. Make necessary changes Remove pathways not found in My species glycogen biosynthesis C4 photosynthesis caffeine biosynthesis Edit pathways operating via a different route Phenylalanine biosynthesis in bacteria vs. My species

19. Make necessary changes AraCyc Pathway: phenylalanine biosynthesis Edit pathways operating via a different route

20. Increase the data content Add pathways from My species not present in the reference database Secondary metabolites Add additional compounds, reactions, and enzymes from My species that cannot be put in a known pathway Write new summaries or revise imported summaries for pathways

21. Provide evidence codes Evidence codes are used for: Pathways Enzyme activities General types of support EV-EXP -> experimental EV-COMP -> computational EV-IC -> inferred by a curator EV-AS -> author statement Additional information about evidence can be captured: EV-EXP-IDA-PURIFIED-PROTEIN Inferred from direct assay EV-EXP-IMP-REACTION-BLOCKED Inferred from mutant phenotype EV-COMP-AINF Inferred by computation automatically without human oversight

22. Provide evidence codes Enzyme activity assays Radiotracer experiments

23. Wed love to have you as part of our Plant Metabolic Network of experts and friends! Add your database to the PMN The PMN can host it as a separate individual species or multi-species database Enzymes from your validated pathways can be incorporated into PlatCyc pathways Newly curated pathways from your species can be added to PlantCyc Pathways Enzyme activities... but you dont have to wait to make a whole new database!

24. Please help us to improve the PMN Review existing pathways Volunteer to go over a set of pathways in your area of expertise E-mail us any time you see a problem Use our feedback form curator@plantcyc.org

25. Please give us advice and information

26. Provide new data Submit any information concerning: Pathways Enzymes Reactions Compounds

27. Please send us new information

28. We are here to help: www.plantcyc.org Please use our data Please use our tools Please help us to improve our databases! Please contact us if we can be of any help! Make an appointment to meet with me during my visit (Puedo tratar de hablar en español) curator@plantcyc.org www.plantcyc.org

29. PMN Acknowledgements Current Curators: - A. S. Karthikeyan (curator) Recent Past Contributors: - Christophe Tissier (curator) - Hartmut Foerster (curator) Collaborators: - Peter Karp (SRI) - Ron Caspi (SRI) - SRI Tech Team - Lukas Mueller (SGN) - Anuradha Pujar (SGN) - Gramene and MedicCyc Peifen Zhang (Director) Sue Rhee (PI) Eva Huala (Co-PI) Tech Team Members: - Bob Muller (Manager) - Larry Ploetz (Sys. Administrator) - Raymond Chetty - Anjo Chi - Vanessa Kirkup - Cynthia Lee - Tom Meyer - Shanker Singh - Chris Wilks

30. We are here to help: www.plantcyc.org Please use our data Please use our tools Please help us to improve our databases! Please contact us if we can be of any help! Make an appointment to meet with me during my visit (Puedo tratar de hablar en español) curator@plantcyc.org www.plantcyc.org