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Mapping file (Excel file) MapMan: a la carte painting of profiling data onto a template of your own design Transcript Scavenger Module 35 ‚BINS‘ - major.

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Presentation on theme: "Mapping file (Excel file) MapMan: a la carte painting of profiling data onto a template of your own design Transcript Scavenger Module 35 ‚BINS‘ - major."— Presentation transcript:

1 Mapping file (Excel file) MapMan: a la carte painting of profiling data onto a template of your own design Transcript Scavenger Module 35 ‚BINS‘ - major functional categories - subdivided into hierarchical subBINS 23,000 genes Image Annotator Module Oliver Thimm Oliver Blaesing Yves Gibon Axel Nagel Svenya Meyer Peter Krüger Joachim Selbig Experimental data, e.g 22k Affy chip, (Excel file) A sketch of your favorite bit of biology (bmp file) Automatic display onto your sketch

2 - Import existing categorised genes 1310 entries 2812 entries Assignment to BINS - Screen Tigr3 gene descriptions with text search algorithms using Kegg enzyme and alternative names keywords user defined keywords 3080 entries 16213 entries Assignment to BINS - Manual correction of binned gene annotations master file Manually corrected file 21918 entries 19584 unique Sorting genes into (rubbish) BINS, with the SCAVENGER module

3 Excerpt from a mapping file BIN 3 is expanded to show its sub-BINS Sub-BIN 3.1 (raffinose metabolism) is expanded to the level of single genes. - identified by a ‚unique identifier (Affy number blablablablabla_at) - will carry the numeric code ‚3.1‘ A mouse over action revealss the assignment, illustrated for 249804_at (encodes galactinol synthase, as is displayed at the bottom of the screen) The mapping file (i) Re-organises the experimental data file. - Groups together all the genes that have a particular numeric code (i.e., are in the same BIN/sub-BIN). (ii) Provides the code via which the resulting sets of data are painted onto your pretty diagrams

4 The User takes a diagram of his/her choice, and decides what data is displayed, where, and how.... Mapping files What data do you want to paint here ? Type in BIN identifier How do you want to show it ? Data files + Frequency histogramm presentation default : shows each gene Mouse over maps Blue = increase Red = decrease Grey = no call White = no change Mouse click at a selected position on the picture

5 Mitochondrial electron transport and ATP synthesis Glycolysis Amino acid synthesis Amino acid breakdown Nucleotides Pectin- esterases XETs, Expansins UDP-sugar metabolism Cellu- lases Syn- thesis Break- down Calvin cycle Syn- thesis Break- down Cellulose and hemicellulose synthesis Cell wall breakdown Hemi- cellose Pectin lyases, polygalacturonases FA synthesis Desaturation TAGTAG Phospho- and galacto- lipids Sterols and sulpholipid Lipid breakdown GDSL lipisaes lipases ( lyso)phos pholipase s beta oxidation gluconeogenesis Glyoxalate cycle Other organic acid trans- formation s Nitrate reduction Ammonium assimilation Ammonium release GABA metabolis m Asp AT Ala AT Branched chain amino acid AT Synthesis Breakdown Salvage Phosphotransfer pyrophosphases Deoxy- nucleotide Pro Arg Hyp Asn Thr Ileu Met Lys Homoserin e synthesis Val Leu Val Ser Gly Cys Phe Tryp Tyr Chorismate synthesis His C-1 metabolism Ascorbate, Glutathione Tetrapyrrole Fermentation Photo- respiration Carbonic anhydrases Raffi- nose Treh- alose Sugar alcohol My o- Ins Misc Call ose. Misc. kin- ases Example of a ‚map‘

6 Extension of the night for 2h, 4h,6h,and 8h Transcripts

7 synthesis pectin lyases polygalacturonases * Pectin *esterases XET‘s Expansins Break down Selective changes of expression of genes involved in cell wall synthesis, breakdown and modification Transcripts

8 2h ext night 2 h into the extended night A few genes induced No genes repressed Painting at the pathway level

9 4h ext night 4 h into the extended night Some genes for glycolytic enzymes are being repressed Induction of two PFP family members and cytosolic FBPase Tho‘ many genes for pyruvate kinases are slightly repressed, one is induced

10 6h ext night 6 h into the extended night Some genes for glycolytic enzymes are being repressed Reciprocal changes of specific PFK/PFP and FBP family members Many genes for pyruvate kinases are slightly repressed, but three are is induced

11 8h ext night 8 h into the extended night - Repression becomes stronger and spreads to more genes Reciprocal changes of specific PFK/PFP and FBP family members Many genes for pyruvate kinases are slightly repressed, but one is induced

12 Cellular functions in an extended night after 2h, 4h,6h,and 8h Looking for changes in general functions

13 23.1 Synthesis 23.2 Breakdown Nucleotide metabolism Lipid metabolism 11.1 FA synthesis 11.2 FA desaturatio n 11.4 Phospho- & galactolipid synthesis 11.9.2 Lipases 11.9.3 (lyso)-phos- pholipases 11.9.4 ß-oxidation 10.2 + 10.3 Cellulose & hemicellulose synthesis 10.6.2 Matrix breakdown 10.6.3 Pectin lyases Polygalacturonases 10.7 XET‘s Expansins 10.5 Pectin *esterases Cell wall Sugars regulate expression of 100’s of genes - Central carbon metabolism - Nitrate, ammonium, amino acid and nucleotide metabolism - RNA and protein synthesis - Lipid metabolism - Cell wall metabolism and modification - Secondary metabolism - Hormone synthesis and sensing - Signalling pathways - Transcription factors

14 Integration with further experiments - comparison with other nutrient challenges Arabidopsis grown in liquid culture under continuous light with full nutrients, or with one limiting... Treatments: Resupply 3mM sucrose to sugar- depleted seedlings. Resupply 3mM nitrate to nitrate- depleted seedlings. Resupply 3mM phosphate to phosphate-depleted seedlings Resupply 3mM sulphate to sulphate-depleted seedlings Overlay plots

15 Overlay the responses to identify similarities and differences on a gene-to-gene basis pgm at end of night vs. WT at end of night 6h Extended night vs. End of normal night Increase by > 1.0 in both Increase by > 0.5 in both Change by < 0.5 in both Decrease by > 0.5 in both Decrease by > 1.0 in both Opposing Responses Mathematical correlation plot for all the genes in pgm vs. in Extended Night Paint out the result for each individual gene onto the map

16 Direct overlay of the responses: 6h extended night vs. pgm Light harvesting proteins etc.

17 carbon and nitrate Overlay plot comparing the response of metabolism to carbon and nitrate deficiency Shared: Induce many genes for amino acid breakdown Shared: Repress - Pectinesterases - Expansins, - XET‘s - PolyGalUAases Many non-shared responses

18 Overlay plot comparing the response of metabolism to nitrate and phosphate deficiency Many shared responses Repression of photosynthesis Repression of chlorophyll synthesis Induction of phenylpropanoid and flavanoid metabolism

19 Overlay plot comparing the response of metabolism to nitrate and sulphate deficiency Many shared responses Repression of photosynthesis Repression of chlorophyll synthesis Induction of phenylpropanoid and flavanoid metabolism

20 Statistical treatment of the responses of different BINS - rigorous support for conclusions - data condensation to aid compariosn of different treatments - support automatic identification of co-regulated processes Display clusters or other statistictically-generated groupings of genes Use to organise and display data sets from crop plants Visualisation of metabolite profiling data sets Combination of data-sets at different levels Adapt display to give information about the expression level as well as the change of expression Extend to visualise the expression of a small set of genes-of-interest across a large collection of arrays for different developmental stages, organs, or treatments. Current Work Ongoing correction of assignments by mining public sites Establish pipelines to import updates of annotation, and expert advice

21 PCA Analysis of transcript profiles: - diurnal cycles in wildtype (biological triplicates) - extended night in wildtype (biological duplicates) - diurnal cycles in pgm (biological duplicates) Wildtype Extended night Wildtype Light Wildtype Dark pgm Component 1: treatments lie on an axis from high to low sugar total of 40 arrays in 20 conditions

22 Correlation plot for ~14,000 genes between Weightings of transcripts in the first principle component and Changes of transcripts in the first 3 hrs after adding sugar to sugar-starved seedlings in liquid culture Response of starved seedlings to added sugar: starved of sugar 3hrs after adding sugar Weighting in the 1 st component, in PCA of diurnal rhythms and extended night treatments

23 1 st component for diurnal changes and extended night Changes after adding sugar to starved seedlings Reciprocal plot Several cell wall degrading enzymes Several TPS‘s Amino acid break- down GDH1 GDH2 ASN1 SO 4 assimi -lation Amino acid synthesis Phenyl- propanoid and flavonoid metabolism Several cell wall degrading enzymes Several TPS‘s Amino acid break- down GDH1 GDH2 ASN1 SO 4 assimi -lation Amino acid synthesis Phenyl- propanoid and flavonoid metabolism

24 MapMan: a la carte painting of profiling data onto a template of your own design Transcript Scavenger Module 35 ‚BINS‘ - major functional categories - subdivided into hierarchical subBINS 23,000 genes Image Annotator Module Oliver Thimm Oliver Blaesing Yves Gibon Axel Nagel Svenya Meyer Peter Krüger Joachim Selbig Mapping file Experimental data, e.g 22k Affy array, as an Excel file A sketch of your favorite bit of biology –bmp file Automatic display onto your sketch Metabolite Scavenger Module 35 ‚BINS‘ - major functional categories - subdivided into hierarchical subBINS 100‘s of metabolites Experimental data, e.g GC-MS profile, as an Excel file

25 L. penelli metabolite profile painted out with Mapman Changes are shown in false color relative to L. esculentum In work

26 An introgression line with improved nitrogen assimilation.... In work Changes are shown in false color relative to L. esculentum

27 An introgression line with improved amino acid biosynthesis.... In work Changes are shown in false color relative to L. esculentum

28 An introgression line with high levels of stress-related metabolites In work Changes are shown in false color relative to L. esculentum

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33 30 min Reprogrammning of metabolism after adding nitrate back to deficient seedlings in liquid culture Induce nitrate reduction Induce OPP cycle Induce sucrose breakdown Induce glycolysis and the TCA cycle

34 Responses can be resolved to the pathway level TCA cycle Mitochondrial electron transport & ATP synthesis Induce specific members of the families for each enzyme needed to move carbon from the end of glycolysis through to 2- oxoglutarate –the acceptor for ammonium Repress non-phosphory- lating NADH dehydrogenases Repress enzymes for gluconeogenesis

35 30 min Reprogrammning of metabolism after adding nitrate back to deficient seedlings in liquid culture 3 hours Induce nitrate reduction Induce OPP cycle Induce sucrose breakdown Induce sulphate reduction Induce nucleotide synthesis and salvage Induce amino acid synthesis Induce several XET‘s and expansins Induce several FA desaturases Inhibit amino acid and nucleotide breakdown Why is Phe synthesis inhibited Inhibition of phenylpropanoid and flavonoid metabolism Induce glycolysis and the TCA cycle Induce photosynthesis

36 Nitrate readdition leads to major changes of transcripts for large enzyme families involved in biosynthetic and secondary metabolism after 30 min,3 hoursand 2 days Very widespread changes Many glucosyltransferases are repressed

37 Cellular responses to nitrate resupply.. after 30 min... and 3 hours

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