Download Document

MapMan: a la carte painting of profiling data
onto a template of your own design
Transcript Scavenger Module
Oliver Thimm
Oliver Blaesing
Yves Gibon
Axel Nagel
Svenya Meyer
Peter Krüger
Joachim Selbig
23,000 genes
35 ‚BINS‘ - major functional categories subdivided into hierarchical subBINS
Mapping file (Excel file)
Experimental data,
e.g 22k Affy chip,
(Excel file)
Image Annotator
Module
Automatic display
onto your sketch
A sketch of your
favorite bit of
biology
(bmp file)
Sorting genes into (rubbish) BINS,
with the SCAVENGER module
- Import existing categorised genes
1310 entries
Assignment
2812 entries
to BINS
- Screen Tigr3 gene descriptions with text search algorithms
using Kegg enzyme
3080 entries
and alternative names
Assignment
16213 entries
keywords
user defined keywords
to BINS
- Manual correction of binned gene annotations
master
file
21918 entries
Manually corrected file
19584 unique
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‘
The mapping file
(i) Re-organises the experimental data file.
- Groups together all the genes that have a
A mouse over action revealss
particular numeric code (i.e., are in the
the assignment, illustrated for
same BIN/sub-BIN).
249804_at (encodes galactinol
(ii) Provides
synthase,
theascode
is displayed
via which
at the resulting sets
ofbottom
data are
ofpainted
the screen)
onto your pretty diagrams
The User takes a diagram of his/her choice, and
decides what data is displayed, where, and how ....
Data
files
Mouse click at a
selected position
on the picture
Frequency
histogramm
presentation
What data do you
want to paint here ?
Type in
BIN identifier
maps
+
Mapping
files
Blue = increase
Red = decrease
Grey = no call
White = no change
default :
shows each
gene
Mouse
over
How do you
want to show it ?
Synthesis
Breakdown
(lyso)phos beta
pholipase oxidation
Glyoxalate
cycle
Calvin cycle
Lipid breakdown
Pro
Arg
Ileu
Met
Leu
Ser
Gly
Tyr
Cys
Tryp
Pro
Arg
Homoserin
e synthesis
Amino acid synthesis
Fermentation
Asn
Val
Thr
Ileu
Example of a ‚map‘
Mitochondrial electron
transport and ATP
synthesis
Carbonic anhydrases
Ammonium
Nitrate
assimilation
reduction
Ammonium release
Ser
Phe
Gly
Tyr
His
Cys Tryp
Leu
Hyp
Met
Lys
C-1 metabolism
Synthesis
Lys
Val
Chorismate
synthesis
Hyp
Asn
Thr
GABA
metabolis Asp
AT
m
Phe
His
Photorespiration
Other
organic
acid
transformation
Brancheds
Ala AT chain amino
acid AT
s
Tetrapyrrole
Ascorbate,
Glutathione
Salvage
Breakdown
Desaturation FA synthesis
lipases
Sterols
and
sulpholipid
Breakdown
Glycolysis
G
Hemicellose
TA
Synthesis
Pectin lyases,
polygalacturonases
Cell wall breakdown
GDSL
lipisaes
Phosphoand
galactolipids
UDP-sugar metabolism
Cellulases
XETs,
Expansins
Cellulose and
hemicellulose
synthesis
Misc.
kinases
Misc Call
ose
.
gluconeogenesis
Pectinesterases
Raffi- Treh- Sugar My
nose alose alcohol oIns
Deoxynucleotide
Phosphotransfer
pyrophosphases
Amino acid breakdown
Nucleotides
Extension of the night for 2h, 4h, 6h, and 8h
Transcripts
Pectin *esterases
synthesis
pectin lyases
polygalacturonases
XET‘s
Expansins
Break
down
Selective changes of expression of genes involved in
cell wall synthesis, breakdown and modification
Transcripts
Painting at the
pathway level
2h ext night
2 h into the
extended night
A few genes induced
No genes repressed
4h ext night
Induction of two PFP family
members and cytosolic FBPase
4 h into the
extended night
Some genes for
glycolytic enzymes
are being repressed
Tho‘ many genes for
pyruvate kinases are
slightly repressed,
one is induced
6h ext night
Reciprocal changes of specific
PFK/PFP and FBP family members
6 h into the
extended night
Some genes for
glycolytic enzymes
are being repressed
Many genes for
pyruvate kinases are
slightly repressed, but
three are is induced
8h ext night
Reciprocal changes of specific
PFK/PFP and FBP family members
8 h into the
extended night Repression
becomes stronger
and spreads to
more genes
Many genes for
pyruvate kinases are
slightly repressed, but
one is induced
Cellular functions in an extended night
after 2h, 4h, 6h, and 8h
Looking for changes
in general functions
Sugars regulate expression
of 100’s of genes
Nucleotide
metabolism
23.1
Synthesis
23.2
Breakdown
11.4 Phospho- &
11.2 FA
11.1 FA
galactolipid synthesis desaturatio synthesis
n
Lipid
metabolism
10.5 Pectin
*esterases
Cell
wall
10.7
XET‘s
Expansins
11.9.2
11.9.3 11.9.4
Lipases (lyso)-phos- ß-oxidation
pholipases
10.2 + 10.3
Cellulose &
hemicellulose
synthesis 10.6.2
Matrix breakdown
10.6.3
Pectin lyases
Polygalacturonases
- 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
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 sugardepleted seedlings.
Resupply 3mM nitrate to nitratedepleted seedlings.
Resupply 3mM phosphate to
phosphate-depleted seedlings
Resupply 3mM sulphate to
sulphate-depleted seedlings
Overlay plots
Overlay the responses to identify
similarities and differences
on a gene-to-gene basis
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
6h Extended night
vs.
End of normal night
Opposing Responses
Comparison of pgm at end of night
vs 6 h extended night
in pgm
vs.
in Extended Night
8
pgm at end of night
vs.
WT at end of night
log2 ratio 6 h XN/ wt EN
6
4
2
0
-2
-4
pgm white vs XN white
pgm dblue vs XN dblue
pgm dred vs XN dred
pgm lblue vs XN lblue
pgm lred vs XN lred
pgm vs XN grey
-6
-8
-8
-6
-4
-2
0
2
4
6
8
log2 ratio pgm EN/wt EN
Mathematical
correlation plot
for all the genes
Paint out the result
for each individual
gene onto the map
Direct overlay of the responses: 6h extended night vs. pgm
Light harvesting
proteins etc.
Overlay plot comparing the response of
Many
metabolism to carbon and nitrate deficiency non-shared
responses
Shared: Repress
- Pectinesterases
- Expansins,
- XET‘s
- PolyGalUAases
Shared: Induce many genes for
amino acid breakdown
Overlay plot comparing the response of
Many
metabolism to nitrate and phosphate deficiency shared
responses
Repression of
photosynthesis
Repression of
chlorophyll
synthesis
Induction of phenylpropanoid
and flavanoid metabolism
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
Current Work
Ongoing correction of assignments by mining public sites
Establish pipelines to import updates of annotation, and expert advice
Display clusters or other statistictically-generated groupings of genes
Visualisation of metabolite profiling data sets
Combination of data-sets at different levels
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
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.
Use to organise and display data sets from crop plants
PCA Analysis of transcript profiles:
- diurnal cycles in wildtype (biological triplicates)
- extended night in wildtype (biological duplicates)
- diurnal cycles in pgm (biological duplicates)
Wildtype
Dark
Wildtype
Extended
night
Wildtype
Light
pgm
Component 1: treatments lie on an axis from high to low sugar
total of 40 arrays in 20 conditions
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
3hrs after
sugar
adding sugar
Weighting in the 1st component,
in PCA of diurnal rhythms and extended night treatments
Changes after adding sugar to
starved seedlings
Several
TPS‘s
Several
cell wall
degrading
enzymes
SO4
assimi
-lation
Reciprocal plot
Phenylpropanoid
and
flavonoid
metabolism
ASN1
Amino acid
synthesis
Amino
acid
breakdown
GDH1
GDH2
1st component for diurnal
changes and extended night
Several
TPS‘s
Phenylpropanoid
and
flavonoid
metabolism
Several
cell wall
degrading
enzymes
ASN1
Amino acid
synthesis
SO4
assimi
-lation
Amino
acid
breakdown
GDH1
GDH2
MapMan: a la carte painting of profiling data
onto a template of your own design
Transcript
MetaboliteScavenger
ScavengerModule
Module
Oliver Thimm
Oliver Blaesing
Yves Gibon
Axel Nagel
Svenya Meyer
Peter Krüger
Joachim Selbig
23,000 genes
100‘s of metabolites
35 ‚BINS‘ - major functional categories 35 ‚BINS‘ - major functional categories subdivided into hierarchical subBINS
subdivided into hierarchical subBINS
Mapping file
Experimental data,
Experimental data,
e.g 22k Affy array,
e.g GC-MS profile,
as an Excel file
as an Excel file
Image Annotator
Module
Automatic display
onto your sketch
A sketch of your
favorite bit of
biology –bmp file
L. penelli metabolite profile painted out with Mapman
Changes are shown in false color relative to L. esculentum
An introgression line with improved nitrogen assimilation ....
Changes are shown in false color relative to L. esculentum
An introgression line with improved amino acid biosynthesis....
Changes are shown in false color relative to L. esculentum
An introgression line with high levels of stress-related metabolites
Changes are shown in false color relative to L. esculentum
Reprogrammning of metabolism
after adding nitrate
Induce
Induce
sucroseculture
back to deficient seedlings in liquid
breakdown
OPP cycle
Induce
glycolysis and
the TCA cycle
Induce
nitrate
reduction
30 min
Responses can be resolved to the pathway level
Repress enzymes
for gluconeogenesis
TCA cycle
Repress
non-phosphorylating NADH
dehydrogenases
Induce specific members
of the families for each
enzyme needed to move
carbon from the end of
glycolysis through to 2oxoglutarate –the
acceptor for ammonium
Mitochondrial electron
transport & ATP synthesis
Reprogrammning of metabolism
after adding nitrate
Induce
Induce
backseveral
to deficient
seedlings
in sucrose
liquid culture
Induce
Induce
several
XET‘s and
expansins
FA
desaturases
breakdown
OPP cycle
Induce
photosynthesis
Induce
glycolysis and the
TCA cycle
Inhibition of
phenylpropanoid
and flavonoid
metabolism
Why is Phe
synthesis
inhibited
Induce
nitrate
reduction
Induce
sulphate
reduction
3 hours
30
min
Induce amino
acid synthesis
Inhibit amino acid and
nucleotide breakdown
Induce
nucleotide
synthesis
and salvage
Nitrate readdition leads to major changes of transcripts for
large enzyme families involved in biosynthetic and
secondary metabolism after 30 min, 3 hours and 2 days
Many
glucosyltransferases
are repressed
Very widespread changes
Cellular responses to nitrate resupply
.. after 30 min ... and 3 hours