El PMN:Tu amigo en el metabolismo

de plantas

Kate Dreher

curator

PMN/AraCyc/TAIR

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)

Choose a database . . .

Use the basic search tools

Finding the information you want . . .

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)?

Basic searching: practice problems 1. What are some of the pathways that talk about “salt stress” in the PMN?

Arabidopsis thaliana proline biosynthesis IMeasurement 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 ISummers93: 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"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"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... 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

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?

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 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

Advanced searching

Advanced Query Form

Downloading the whole database

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?

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?

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 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

OMICs viewer

Do your experiment (or get your data)

Create your input file

Upload and analyze your data Absolute values Time course Relative values

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?

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

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

Validating your own metabolic database

PathoLogic errs on the side of over-prediction

Curators / Scientists validate pathways . . .

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?”

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

Make necessary changes

AraCyc Pathway: phenylalanine biosynthesis

Edit pathways operating via a different route

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

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

Provide evidence codes

Enzyme activity assays

Radiotracer experiments

We’d love to have you as part of ourPlant 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 don’t have to wait to make a whole new database!

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

Please give us advice and information

Please give us advice and information

Provide new data

Submit any information concerning: Pathways Enzymes Reactions Compounds

Please send us new information

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

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

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